Efficient Distribution of a Novel Zirconium-89 Labeled Anti-cd20 Antibody Following Subcutaneous and Intravenous Administration in Control and Experimental Autoimmune Encephalomyelitis-Variant Mice

Abstract

Objective: To investigate the imaging and biodistribution of a novel zirconium-89 (89Zr)-labeled mouse anti-cd20 monoclonal antibody (mAb) in control and experimental autoimmune encephalomyelitis (EAE) mice following subcutaneous (s. c.) and intravenous (i.v.) administration. Background: Anti-cd20-mediated B-cell depletion using mAbs is a promising therapy for multiple sclerosis. Recombinant human myelin oligodendrocyte glycoprotein (rhMOG)-induced EAE involves B-cell-mediated inflammation and demyelination in mice. Design/Methods: C57BL/6J mice (n = 39) were EAE-induced using rhMOG. On Day 14 post EAE induction, 89Zr-labeled-anti-cd20 mAb was injected in control and EAE mice in the right lower flank (s.c.) or tail vein (i.v.). Positron emission tomography/computed tomography (PET/CT) imaging and gamma counting (ex vivo) were performed on Days 1, 3, and 7 to quantify tracer accumulation in the major organs, lymphatics, and central nervous system (CNS). A preliminary study was conducted in healthy mice to elucidate full and early kinetics of the tracer that were subsequently applied in the EAE and control mice study. Results:89Zr-labeled anti-cd20 mAb was effectively absorbed from s.c. and i.v. injection sites and distributed to all major organs in the EAE and control mice. There was a good correlation between in vivo PET/CT data and ex vivo quantification of biodistribution of the tracer. From gamma counting studies, initial tracer uptake within the lymphatic system was found to be higher in the draining lymph nodes (inguinal or subiliac and sciatic) following s.c. vs. i.v. administration; within the CNS a significantly higher tracer uptake was observed at 24 h in the cerebellum, cerebrum, and thoracic spinal cord (p < 0.05 for all) following s.c. vs. i.v. administration. Conclusions: The preclinical data suggest that initial tracer uptake was significantly higher in the draining lymph nodes (subiliac and sciatic) and parts of CNS (the cerebellum and cerebrum) when administered s.c. compared with i.v in EAE mice.

Keywords: biodistribution; experimental autoimmune encephalomyelitis; intravenous; monoclonal antibody; neuroimaging; positron emission tomography imaging; radiolabeling; subcutaneous.

Copyright © 2019 Migotto, Mardon, Orian, Weckbecker, Kneuer, Bhalla and Reutens.

Figures

Figure 1

Study design. aC57BL/6 mice post-EAE induction who had reached the peak of the disease on Days 14–15. bControl mice were sham-injected (i.e., subjected to the same procedure as EAE-induced mice, except that rhMOG was replaced with saline). cWhole body clearance and biodistribution of the tracer were assessed by PET/CT imaging. dOrgans excised from a subset of mice (n = 7–9) and assessed for biodistribution of the tracer by gamma counting. EAE, experimental autoimmune encephalomyelitis; MBq, megaBecquerel; n, number of mice; PET/CT, positron emission tomography/computed tomography; rhMOG, recombinant human myelin oligodendrocyte glycoprotein.

Figure 2

The EAE mean clinical score and percent change in weight in EAE and control mice at various time points (n = 39). Data presented as mean ± SD. EAE, experimental autoimmune encephalomyelitis; i.v., intravenous; s.c., subcutaneous; SD, standard deviation.

Figure 3

Whole body clearance of the 89Zr-labeled anti-CD20 mAb following s.c. and i.v. injection in control and EAE mice (n = 5–9 mice per time point) measured using PET/CT. Data presented as mean ± SEM. i.v., intravenous; mAb, monoclonal antibody; n, number of mice; s.c., subcutaneous; SEM, standard error of the mean 89Zr, Zirconium-89.

Figure 4

Comparison of PET/CT in vivo biodistribution (A–D) and in vivo imaging (E,F) of the 89Zr-labeled anti-CD20-mAb in control and EAE mice following s.c. and i.v. injection. (A) Biodistribution of the tracer following s.c. injection in control mice (n = 3–6). (B) Biodistribution of the tracer following s.c. injection in EAE mice (n = 3–9). (C) Biodistribution of the tracer following i.v. injection in control mice (n = 1–2)#. (D) Biodistribution of the tracer following i.v. injection in EAE mice (n = 3–4). (E)In vivo imaging following s.c. injection of the tracer in control and EAE mice. (F)In vivo imaging following i.v. injection of the tracer in control and EAE mice. Data presented as mean ± SEM. #Sample size was very low (n = 1 or 2) to calculate the SEM values. % ID/g, percentage injected dose per gram; EAE, Experimental Autoimmune Encephalomyelitis; i.v., intravenous; mAb, monoclonal antibody; n, number of mice; PET/CT, positron emission tomography/computed tomography; s.c., subcutaneous; SEM, standard error of the mean; 89Zr, Zirconium-89.

Figure 5

Comparison of gamma counter biodistribution of the 89Zr-labeled anti-CD20 mAb in control and EAE mice following s.c. (A,B) and i.v. (C,D) injection. (A) Biodistribution of the tracer following s.c. injection in control mice (n = 7). (B) Biodistribution of the tracer following s.c. injection in EAE mice (n = 9). (C) Biodistribution of the tracer following i.v. injection in control mice (n = 1–3). (D) Biodistribution of the tracer following i.v. injection in EAE mice (n = 3–4). Data presented as mean ± SEM. % ID/g, percentage injected dose per gram; EAE, experimental autoimmune encephalomyelitis; i.v., intravenous; LNs, lymph node; mAb, monoclonal antibody; n, number of mice; s.c., subcutaneous; SEM, standard error of the mean; 89Zr, Zirconium-89.

Figure 6

Comparison of gamma counter biodistribution of the 89Zr-labeled anti-CD20 mAb in control and EAE mice across specific LNs following s.c. (A,B) and i.v. (C,D) injection. (A) Biodistribution of the tracer in specific LNs following s.c. injection in control mice (n = 7). (B) Biodistribution of the tracer in specific LNs following s.c. injection in EAE mice (n = 9). (C) Biodistribution of the tracer in specific LNs following i.v. injection in control mice (n = 1–2)#. (D) Biodistribution of the tracer in specific LNs following i.v. injection in EAE mice (n = 3–4). Data presented as mean ± SEM. #Sample size was very low (n = 1 or 2) to calculate the SEM values for Iliac LN, Sciatic LN, mandibular LN on Day 1 and for deep cervical LN on Day 3 following i.v. injection in control mice. A 2-way analysis of variance (ANOVA) test was applied to detect significant association between tracer uptake and EAE. % ID/g, percentage injected dose per gram; EAE, experimental autoimmune encephalomyelitis; i.v., intravenous; LLOD, lower limit of detection (less than 3x background signal); LNs, lymph nodes; mAb, monoclonal antibody; n, number of mice; s.c., subcutaneous; SEM, standard error of the mean; 89Zr, Zirconium-89.

Figure 7

Comparison of gamma counter biodistribution of the 89Zr-labeled anti-CD20 mAb in control and EAE mice across CNS following s.c. (A,B) and i.v. (C,D) injection. (A) Biodistribution of the tracer in the CNS following s.c. injection in control mice (n = 4). (B) Biodistribution of the tracer in the CNS following s.c. injection in EAE mice (n = 9). (C) Biodistribution of the tracer in the CNS following i.v. injection in control mice (n = 1–2)#. (D) Biodistribution of the tracer in CNS following i.v. injection in EAE mice (n = 4–5). *p < 0.05, using a Chi-squared test of independence (for s.c.: χ2 [1, N = 39] = 6.6–22.5, α = 0.05; i.v.: c2 [1, N = 15] = 5.0–16.16, p < 0.05) to compare CNS biodistribution of the tracer in control and EAE mice, highlighting significant differences in the relationship between CD20 antibody uptake and EAE. ∧p < 0.05, using single factor ANOVA [F(1) > 4.84; p < 0.05] and independent t-test to compare CD20 antibody uptake in EAE mice following s.c. and i.v. administration, suggesting regional early preferential CNS uptake in EAE following s.c. administration. Data presented as mean ± SEM. #Sample size was too low (n = 1 or 2) to calculate the SEM values for lumbar spinal cord, medulla/pons, and cerebellum on Day 1 following i.v. injection in control mice. % ID/g, percentage injected dose per gram; Ab, antibody; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; i.v., intravenous; LLOD, lower Limit of detection (less than 3x background signal); mAb, monoclonal antibody; N, total number of mice; n, number of mice; s.c., subcutaneous; SEM, standard error of the mean; 89Zr, Zirconium-89.