Ex-vivo delivery of monoclonal antibody (Rituximab) to treat human donor lungs prior to transplantation.
BACKGROUND
Ex-vivo lung perfusion (EVLP) is an innovative platform for assessing donor lungs in the pre-transplant window. In this study, we demonstrate an extension of its utility by administering the anti-CD20 monoclonal antibody, Rituximab, during EVLP. We hypothesized that this would lead to targeted depletion of allograft B-cells which may provide significant clinical benefit, including the potential to reduce latent Epstein-Barr virus (EBV) and decrease the incidence of post-transplant lymphoproliferative malignancies.


METHODS
Twenty human donor lungs rejected for transplantation were placed on EVLP with (n = 10) or without (n = 10) 500 mg of Rituximab. Safety parameters such as lung physiology and inflammatory cytokines were evaluated. We measured the delivery efficacy through flow cytometry, immunohistochemistry and ELISA. An in-vitro culture assay, in the presence of complement, was further conducted to monitor whether B-cell depletion would occur in Rituximab-perfused samples.


FINDINGS
Rituximab was successfully delivered to human lungs during EVLP as evidenced by flow cytometric binding assays where lung tissue and lymph node biopsies demonstrated occupied CD20 epitopes after perfusion with the antibody. Lymph nodes from Rituximab perfusions demonstrated a 10.9 fold-reduction in CD20+ staining compared to controls (p = 0.0003). In lung tissue, Rituximab resulted in an 8.75 fold-reduction in CD20+ staining relative to controls (p = 0.0002). This decrease in CD20+ binding illustrates the successful delivery and occupation of epitopes after perfusion with the Rituximab. No apparent safety concerns were seen as exhibited by markers associated with acute cell injury (e.g., proinflammatory cytokines), cell death (e.g., TUNEL staining), or pulmonary physiology. In a post-perfusion tissue culture model, the addition of complement (human serum) resulted in evidence of B-cell depletion consistent with what would be expected with posttransplant activation of bound Rituximab.


INTERPRETATION
Our experiments illustrate the potential of EVLP as a platform to deliver monoclonal antibody therapies to treat donor lungs pretransplant with the goal of eliminating a latent virus responsible for considerable morbidity after lung transplantation.


FUNDING
Supported by the University Health Network Transplant Center.
['Ku|Terrance J Y|TJY|', 'Ribeiro|Rafaela V P|RVP|', 'Ferreira|Victor H|VH|', 'Galasso|Marcos|M|', 'Keshavjee|Shaf|S|', 'Kumar|Deepali|D|', 'Cypel|Marcelo|M|', 'Humar|Atul|A|']
D000911:Antibodies, Monoclonal D001402:B-Lymphocytes D015415:Biomarkers D016207:Cytokines D020031:Epstein-Barr Virus Infections D006801:Humans D018836:Inflammation Mediators D008168:Lung D016040:Lung Transplantation D008212:Lymphocyte Depletion D010477:Perfusion D011300:Preoperative Care D000069283:Rituximab D014019:Tissue Donors
Rituximab Ex-vivo lung perfusion Epstein-Barr virus Monoclonal therapy PTLD