Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis

Abstract

Background: B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE).

Methods: Female C57BL/6 J mice were induced with EAE through immunization with myelin oligodendrocyte glycoprotein (MOG_1-125). PET imaging of naïve and EAE mice was performed 19 h after administration of [⁶⁴Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted.

Results: Radiolabelling of DOTA-conjugated CD19-mAb with ⁶⁴Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/μmol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02 ± 0.092 vs. 1.68 ± 0.06 percentage of injected dose per gram, % ID/g, p = 0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22 ± 0.020 vs. 0.12 ± 0.003 % ID/g, p = 0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry-providing further evidence that [⁶⁴Cu]CD19-mAb enables visualization of B cell infiltration into the CNS of EAE mice.

Conclusion: CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

Keywords: B cells; CD19; EAE mice; Multiple sclerosis; PET.

Fig. 1

Study design showing timeline of disease induction, immunostaining, PET/CT imaging, and ex vivo analyses. Twenty female C57BL/6 J mice were induced with EAE via subcutaneous administration of myelin oligodendrocyte glycoprotein (MOG_1–125) and intraperitoneal injection of pertussis toxin (PTX). Mice were subsequently monitored for signs of paralysis from day 8 onwards. On day 14, n = 6 mice with hindlimb paralysis (scores 2.5–3.5) and n = 5 naive mice were intravenously injected with [⁶⁴Cu]CD19-mAb and imaged using PET/CT 24 h later. The remaining EAE mice were used for in vitro autoradiography/blocking and immunostaining studies

Fig. 2

B cell infiltrates are present in brainstem, meninges, and white matter of EAE mice. Representative B220 immunostaining of naïve and EAE mouse brain tissue (n = 7 EAE, n = 5 naïve mice, average of 4 slices per animal). Scale bars are 5 mm in low magnification (×1) sagittal brain images and 100 μm in high magnification (×20) images of the brainstem, meninges, and cerebellar white matter

Fig. 3

CD19-PET imaging shows increased signal in the brain of EAE mice. a Representative sagittal brain PET/CT images of naïve and EAE mice 24 h after [⁶⁴Cu]CD19-mAb injection. b Quantitation of tracer binding in medulla, pons, white matter, and whole brain, n = 5 per group. *p = < 0.05, **p = < 0.01

Fig. 4

Ex vivo biodistribution confirmed in vivo PET findings. Ex vivo gamma counting of [⁶⁴Cu]CD19-mAb biodistribution in (a) central nervous system and (b) peripheral tissues 24 h after tracer injection, n = 5 per group, *p = < 0.05, **p = < 0.01, ***p = < 0.001, ****p = < 0.0001

Fig. 5

High-resolution ex vivo autoradiography of brain sections and spinal cord tissue further confirmed in vivo PET results. Ex vivo autoradiography (autorad) of (a) 40 μm-thick sagittal brain sections and (b) dissected whole spinal cords—cervical/thoracic (left) and lumbar (right). Nissl staining and photographs are shown for brain sections and spinal cords respectively. (c) Quantitation of radiotracer signal in brainstem, cerebellar white matter (WM), and spinal cord regions, expressed as mean pixel intensity divided by decay-corrected dose injected (MPI/dose); n = 4 per group for brainstem analysis (since brainstem was not successfully dissected for all mice), n = 5 per group for spinal cord analysis. *p = < 0.05, **p = < 0.01, ***p = < 0.001

Fig. 6

Ex vivo autoradiography correlates with PET or B220 immunohistochemistry. Correlation between ex vivo autoradiography and PET or B220 immunohistochemistry of (A/C) cerebellar white matter and (B/D) cerebellum + brainstem (i.e., hindbrain), naïve, and EAE mice (n = 3–4 per group)