Imaging Mass Cytometry and Single-Cell Genomics Reveal Differential Depletion and Repletion of B-Cell Populations Following Ofatumumab Treatment in Cynomolgus Monkeys.

Ofatumumab is the first, fully human, anti-CD20 monoclonal antibody in Phase 3 development for multiple sclerosis (MS). The study focused on changes in lymphocyte subsets in blood and lymphoid tissues and on potential novel biomarkers as a result of anti-CD20 antibody action in Cynomolgus monkeys treated with human equivalent doses of subcutaneous (s.c.) ofatumumab on Days 0, 7, and 14. Axillary lymph nodes (LNs) and blood samples were collected at various time points until Day 90. Lymphocyte subsets were quantified by flow cytometry, while morphological and immune cell changes were assessed by imaging mass cytometry (IMC), immunohistochemistry (IHC), in situ hybridization (ISH), and transcriptome analyses using single-cell methodology. Ofatumumab treatment resulted in a potent and rapid reduction of B cells along with a simultaneous drop in CD20+ T cell counts. At Day 21, IHC revealed B-cell depletion in the perifollicular and interfollicular area of axillary LNs, while only the core of the germinal center was depleted of CD20+CD21+ cells. By Day 62, the perifollicular and interfollicular areas were abundantly infiltrated by CD21+ B cells and this distribution returned to the baseline cytoarchitecture by Day 90. By IMC CD20+CD3+CD8+ cells could be identified at the margin of the follicles, with a similar pattern of distribution at Day 21 and 90. Single-cell transcriptomics analysis showed that ofatumumab induced reversible changes in t-distributed stochastic neighbor embedding (t-SNE) defined B-cell subsets that may serve as biomarkers for drug action. In summary, low dose s.c. ofatumumab potently depletes both B cells and CD20+ T cells but apparently spares marginal zone (MZ) B cells in the spleen and LN. These findings add to our molecular and tissue-architectural understanding of ofatumumab treatment effects on B-cell subsets.

Fingolimod inhibits brain atrophy and promotes brain-derived neurotrophic factor in an animal model of multiple sclerosis.

Longitudinal brain atrophy quantification is a critical efficacy measurement in multiple sclerosis (MS) clinical trials and the determination of No Evidence of Disease Activity (NEDA). Utilising fingolimod as a clinically validated therapy we evaluated the use of repeated brain tissue volume measures during chronic experimental autoimmune encephalomyelitis (EAE) as a new preclinical efficacy measure. Brain volume changes were quantified using magnetic resonance imaging (MRI) at 7 Tesla and correlated to treatment-induced brain derived neurotrophic factor (BDNF) measured in blood, cerebrospinal fluid, spinal cord and brain. Serial brain MRI measurements revealed slow progressive brain volume loss in vehicle treated EAE mice despite a stable clinical score. Fingolimod (1 mg/kg) significantly ameliorated brain tissue atrophy in the cerebellum and striatum when administered from established EAE disease onwards. Fingolimod-dependent tissue preservation was associated with induction of BDNF specifically within the brain and co-localized with neuronal soma. In contrast, therapeutic teriflunomide (3 mg/kg) treatment failed to inhibit CNS autoimmune mediated brain degeneration. Finally, weekly anti-IL-17A antibody (15 mg/kg) treatment was highly efficacious and preserved whole brain, cerebellum and striatum volume. Fingolimod-mediated BDNF increases within the CNS may contribute to limiting progressive tissue loss during chronic neuroinflammation.