A novel CD20-conjugated γδ T cell therapy via unnatural sugar-mediated chemical modification for the treatment of EBV-PTLD

Abstract Background: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication following hematopoietic stem cell and solid organ transplantation. Although CD20-targeted antibodies and EBV-specific T cells have shown promise, many patients remain refractory to these therapies. Gamma delta T (γδ T) cells are emerging as a potent and universal platform for adoptive cell therapy due to their MHC-independent antiviral and antitumor activity. However, their clinical efficacy is limited by poor tumor-targeting efficiency. To address this limitation, conjugating γδ T cells with CD20-specific antibodies may offer a viable therapeutic strategy for EBV-PTLD. Methods: We utilized antibody–cell conjugation (ACC) technology based on metabolic glycan labeling (MGL), which modifies cell-surface sialic acids with bioorthogonal handles, enabling rapid and efficient conjugation of CD20 antibodies to γδ T cell surface sialoglycans via click chemistry. In vitro, the CD20 antibody-conjugated γδ T (CD20-γδ T) cells were co-cultured with EBV and CD20-positive targets, including EBV-transformed lymphoblastoid cell lines (EBV-LCLs), Raji cells, rituximab-resistant Raji cells, and primary EBV-PTLD patient-derived cells. For in vivo evaluation, NPG mice bearing Raji-luciferase tumors were treated intravenously with CD20 antibody, γδ T cells or CD20-γδ T cells every three days for a total of six doses. Mice were monitored for tumor development by bioluminescence imaging, body weight and survival. In vivo kinetics of the infused CD20-γδ T cells were also evaluated. Results: CD20 antibodies were efficiently conjugated to γδ T cell surfaces within 2 hours, achieving ~100% conjugation efficiency, a high cell-surface antibody density (~10⁶ antibodies per γδ T cell), and no detectable impairment of cell function. In vitro experiments showed that CD20-γδ T cells exhibited superior cytotoxicity against CD20+ cell lines and primary EBV-PTLD patient-derived primary cells, compared to CD20 antibody alone or γδ T cells without CD20 antibody conjugation. The in vivo mice model demonstrated that CD20-γδ T cells treatment strongly suppressed the aggressive proliferation of Raji tumor with no observed toxicity, while γδ T cells or CD20 antibody showed moderate inhibition. The median survival time of the tumor-bearing mice were extended from 16 days (untreated) to 20 days (γδ T cells), 28 days (CD20 antibody), and 48 days (CD20-γδ T cells). Conclusions: We developed a novel off-the-shelf CD20-γδ T cell therapy using MGL-based ACC technology, effectively combining the innate antitumor activity of γδ T cells with the precision of CD20 targeting. This approach exhibits potent efficacy against EBV-PTLD in vitro and in vivo, offering a promising allogeneic immunotherapy for refractory EBV-associated diseases.