Evaluation of two novel effectorless immunoPET agents for tracking human CD19-positive B lymphocytes in experimental autoimmune encephalomyelitis

Clinical trials of B cell depletion therapies in multiple sclerosis (MS) have demonstrated promising results for attenuating key symptoms and revolutionized our understanding of B cell involvement in this debilitating disease. Unfortunately, standard clinical imaging methods cannot quantify B cell load in the central nervous system (CNS), limiting our ability to: stratify patients for anti-B cell therapy and monitor therapeutic response. To address these critical needs, we generated two novel positron emission tomography (PET) tracers, one monoclonal antibody (mAb) and one fragment antigen binding (Fab) format, for non-invasive whole-body imaging of human CD19 + B cells. We then assessed their utility in a widely used B cell-driven murine model of MS, experimental autoimmune encephalomyelitis (EAE) expressing human CD19. 64 Cu-hCD19-mAb and 89 Zr-hCD19-Fab were produced with high radiochemical purity (> 99% instant thin layer chromatography [ITLC]) and molar activity (mAb: 296 ± 120, Fab: 26.5 Megabecquerel [MBq]/nmol). Their ability to detect B cells in lymphoid organs and CNS tissues in EAE mice was evaluated using PET, gamma counting, autoradiography, and histology. PET imaging with 64 Cu-hCD19-mAb revealed significantly higher brain signal in EAE compared to naïve mice, whereas no difference was observed with the 89 Zr-hCD19-Fab tracer. Ex vivo autoradiography and gamma counting revealed elevated binding of both tracers in CNS tissues from EAE versus naïve mice; gamma counting showed 50-fold higher absolute binding of the mAb tracer in the brain compared to the Fab. Notably, signal distribution of both tracers in autoradiography brain images matched B220 immunohistochemistry. B cell egress was detected from bone marrow in EAE mice with both tracers, but only the mAb tracer showed decreased signal in blood and spleen. Both tracers were specific for human CD19 + B cells, as shown by significant reductions in spleen-to-blood and brain-to-blood ratios for EAE-blocking and wild-type EAE mice compared to EAE mice expressing human CD19. Our data indicate advantages of the mAb CD19-targeting tracer compared to the Fab version. Specially, the binding signal of 64 Cu-hCD19-mAb reflects alterations in the B cell load in both peripheral and CNS tissues of EAE mice with higher fidelity in vivo and ex vivo compared to the Fab, thus warranting further investigation of 64 Cu-hCD19-mAb in the context of MS pathology.