Novel insights in therapeutic antibody therapies using advanced microscopy techniques Free

Introduction Immunotherapies have shown exceptional potential in achieving deep and durable remissions in Multiple Myeloma (MM). However, the complex interactions between immunoreceptors on MM cells and their surrounding microenvironment are not yet fully understood, presenting a major hurdle in optimizing these therapies. The advent of super-resolution imaging technologies, such as the single-molecule-sensitive methoddirect Stochastic Optical Reconstruction Microscopy (dSTORM) and lattice light-sheet microscopy (LLS), enable precise visualization of receptor organization, clustering, and interaction dynamics. Leveraging these tools, we aim to decode the spatial organization and functional dynamics of immunoreceptors in MM, using CD38 as a model receptor.

Methods We applied dSTORM to map CD38 expression in Daratumumab (Dara) resistant (n=22) and Dara naïve (n=30) MM patients, detecting residual CD38 in CD38 negative patients (n=3) using the multi-epitope (ME) antibody CD38ME. As two-color dSTORM imaging and subsequent colocalization analysis revealed evidence of epitope shielding by therapeutic Dara, we tested whether Isatuximab (Isa) can overcome this effect. We compared the detectable receptors by Dara, Isa or a 1 to 1 mixture, and performed colocalization analysis in AMO1 cells. Additionally, LLS was employed to visualize the CD38 distribution in RPMI8226 cells for 1 hour following therapeutic antibody addition.

Results Using dSTORM we found a significant reduction of detectable CD38 receptors in resistant versus naïve patients (21.7±3.8 vs. 32.1±2.4 clusters/µm², p=0.004). We identified 3 patients as CD38-negative by flow cytometry (FC), but found residual CD38 expression using dSTORM. As the interval between sampling and last Dara treatment was 14 to 24 days, we hypothesized the cause could be epitope shielding by therapeutic Dara still bound to CD38 receptors. Two-color dSTORM images revealed barely any colocalization ranging from 0.6% to 1.3% between CD38ME and Dara. Much more colocalization (7.5% and 7.2%) was found in 2 patients who had last been treated 91 and 468 days before sampling. Together this data confirms epitope shielding by therapeutic Dara for at least 24 days post-treatment. To determine whether this effect can be overcome by treatment with Isa, AMO1 cells stained with Dara, Isa or a 1 to 1 mixture of both were imaged. We detected more CD38 receptors with Dara compared to Isa (44.0±4.7 vs 26.2±3.1 clusters/µm², p=0.03), but their combination yielded no significant difference compared to Dara alone. Colocalization analysis of two-color dSTORM images revealed only 2% of colocalization between Dara and Isa, suggesting that Dara and Isa compete for binding to CD38.

Applying LLS to investigate the CD38 distribution upon therapeutic antibody engagement, we found that CD38 polarization occurred only in 4% of Dara-treated cells within the first 10 minutes, whereas Isa induced polarization more slowly but also more efficiently, reaching 24% after about 25 minutes. We observed that the majority of CD38 clusters localized near the microtubule-organizing center, but no consistent spatial pattern could be identified. Similar results were observed when Dara and Isa were co-incubated. The mechanisms behind these observations remain unclear and need further study.

Conclusion Our study demonstrates the value of dSTORM in overcoming detection limits. We identified residual CD38 expression in patients deemed CD38-negative by FC using CD38ME. Minimal colocalization between Dara and CD38ME was found up to 24 days post-treatment, in contrast with greater colocalization at later time points, suggesting epitope shielding by therapeutic Dara for up to 24 days post-treatment. Additionally, our findings show competitive binding to CD38 by Dara and Isa, hence Isa staining and/or treatment cannot overcome the issue of epitope shielding. By LLS we showed that Dara and Isa affect CD38 spatial distribution similarly, with both antibodies inducing CD38 polarization. However, Isa induces slower but more sustained CD38 polarization compared to Dara, highlighting potential differences in their mechanisms of action. These two imaging approaches combined can provide critical insights into the mechanisms of immune cell-mediated killing as well as mechanisms underlying therapeutic response and resistance, thereby improving diagnostics and enabling personalized treatment strategies for MM patients.