Lattice light-sheet microscopy reveals patient specific responses to CD20-directed antibodies in B cell malignancies Free

Introduction CD20-targeting monoclonal antibodies (mAbs) are in clinical use and standard of care in the treatment of hematological malignancies and autoimmune diseases, including rituximab (RTX), obinutuzumab (OBZ) or ofatumumab (OFA). These agents differ in their structure, epitope specificity and modes of action (MoA), yet the precise molecular basis for their divergent clinical profiles remains incompletely understood. Historically, MoAs have been inferred using biochemical complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays, flow cytometry for B cell depletion kinetics and confocal microscopy to determine cell viability and apoptosis. However, these approaches lack the combined spatial and temporal resolution needed to resolve real-time dynamics at the single cell or molecular level. Here, lattice light-sheet (LLS) microscopy can be used advantageously to visualize in real-time interactions of tumor cells with mAbs reaching high spatiotemporal resolution giving novel insights into binding kinetics, receptor cluster formation and complement deposition. Such dynamic high-resolution insights into antibody-antigen-engagement and downstream immune synapse formation are not achievable with conventional flow cytometry or confocal microscopy, offering a powerful new platform to elucidate and potentially optimize the molecular mechanisms that underlie CD20-directed therapies.

Methods To acquire an interaction profile of cells with components of the immune system, fluorescently labeled mAbs RTX (5µg), OFA (10µg), OBZ (20µg) and 2H7 (10µg, serving as control mAb) were individually applied to Raji cells and primary B cells isolated from anonymized CLL patient samples. Cells were co-incubated with 40µg of the prelabeled complement component 1q (C1q) for 1 hour at 37 °C in RPMI medium, while imaging with an LLS microscope. In a second set of experiments, SiR actin (1:1000) was utilized to visualize mAb-binding induced cytoskeletal changes of B cells. Colocalization of C1q with the respective mAbs and accumulation on the surface of tumor cells was determined over time. To investigate cytoskeletal remodeling, the lengths of membrane protrusions and the degree of actin polarization were measured dynamically during the imaging period.

Results Raji cells co-incubated with OFA and C1q showed a significantly reduced total amount of complex binding compared to RTX and 2H7. The C1q binding to OBZ was also significantly reduced but remained higher than observed with OFA. In actin-stained cells, the total amount of bound complex was reduced for all mAbs, but still highest for RTX. During image acquisition OFA-treated cells maintained stable protrusion lengths, whereas for RTX-treated Raji cells the lengths reduced significantly. This reduction was accompanied by a higher polarization degree observable for all mAbs except OFA. Notably, in our first experiments the total amount of bound complex for each tested mAb showed heterogenic distribution across individual cells and patient samples: In a therapy-naïve CLL patient OFA induced the highest C1q binding events, while OBZ showed the lowest. In contrast, a second therapy-naïve CLL patient displayed the opposite pattern with OBZ inducing strongest C1q binding. In a third patient, who was in first relapse 10 years after RTX-bendamustine therapy, the highest binding was observed for 2H7 whereas OBZ again showed the lowest activity. Until now, clear cytoskeletal changes such as protrusions and polarization have been less pronounced in primary CLL samples compared to Raji cells.

For each condition 20-30 cells were imaged and analyzed.

Conclusion LLS microscopy enables real-time visualization of live-cell interactions between patient derived B cells, therapeutic mAbs and the C1q protein, offering novel insights into inter-individual variability in treatment response. The observed differences in C1q binding and cytoskeletal remodeling suggest that these responses are patient specific and may be influenced by underlying factors such as disease genetics or prior therapies. LLS microscopy holds significant potential to support personalization of immunotherapies in hematologic malignancies by identifying the most effective mAb for each individual patient. When integrated with genetic and molecular profiling, LLS microscopy will further contribute to a broader understanding of underlying mechanisms of organism-treatment interactions.