Ex-Vivo Lung Perfusion Mediated Delivery of Rituximab to Clear Latent Epstein-Barr Virus

Abstract

Purpose The risk of post-transplant lymphoproliferative disorder (PTLD), a B-cell malignancy caused by Epstein-Barr virus (EBV), is highest when latent virus is transmitted from B-cells within the allograft to a naive recipient. Ex-vivo lung perfusion (EVLP) is a potential platform to modify grafts before transplantation. We hypothesized that EVLP-mediated delivery of Rituximab (RTX), an antibody targeting CD20+ B-cells, may safely clear latent EBV from donor lungs and attenuate viral transmission. Methods Rejected donor lungs were placed on EVLP alone (N = 7) or on EVLP with 500mg of RTX (N = 7) for 5-12 hours. Flow cytometry and immunohistochemistry (IHC) were used to quantify the proportion of CD19+ or CD20+ B-cells, and EBV specific proteins and RNA from lung tissue and lymph nodes (LN) collected pre- and post-perfusion. Perfusate was collected to measure proinflammatory cytokines indicative of tissue damage. Results RTX was successfully delivered to lungs with no observable physiologic adverse effects. The ratio of post- to pre-perfusion live B-cells was used to assess the efficacy of RTX-mediated B-cell delivery. Lungs perfused with RTX demonstrated a lower CD20+ cell ratio in treated tissue vs control (median ratio = 0.079 vs 0.58, p = 0.016) (Figure 1). A similar decrease was observed in treated LNs vs control (median ratio = 0.20 vs 0.99, p = 0.016) suggesting that RTX penetrated the graft and coated B-cells. This difference was not apparent among the CD19+ B-cells. IHC results were concordant with these findings. There were no differences in proinflammatory cytokines between groups suggesting the approach is safe. Conclusion Our results suggest efficient delivery and binding of RTX to CD20+ B-cells through EVLP as early as 5 hours post-RTX administration with no observable adverse effects. This study provides evidence that EVLP can effectively serve as a platform for pre-transplant cell depletion and hopefully decrease incidence of PTLD in lung transplant recipients.