Antiviral efficacy of EBV and CMV bispecific cytotoxic t lymphocytes following allogeneic hematopoietic stem cell transplantation

Abstract Purpose: To improve the cure rate of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), it is crucial to reduce treatment-related mortality caused by severe viral infections. Adoptive transfer of virus-specific CTLs (e.g., CMV-CTLs, EBV-CTLs) has proven effective against post-allo-HSCT viral infections. To date, no studies have evaluated bispecific EBV/CMV-targeted cytotoxic T lymphocytes (CTLs) for post-allo-HSCT viral infections. We aimed to investigate the antiviral efficacy of bispecific EBV/CMV-CTLs in allo-HSCT recipients. Methods: We conducted a retrospective analysis of 23 patients who developed EBV or CMV infection after allo-HSCT between October 2020 and May 2025, all of whom received bispecific EBV/CMV-CTLs therapy for antiviral treatment. Key endpoints included treatment response, prophylactic efficacy, safety profile, and overall survival (OS) rate. Results: Twenty-three patients received a total of 28 infusions of bispecific EBV/CMV-CTLs. In terms of treatment response, following the infusion of bispecific CTLs, the peak viral loads of EBV/CMV decreased significantly from 32.5 (range: 1.2-2210.0)×10³ copies/mL to 12.7 (range: 0-213.4)×10³ copies/mL. Following initial bispecific CTL infusion, the cumulative complete response (CR) rates for EBV/CMV infections at weeks 4 and 8 were 56.5% and 78.3%, respectively. The ultimate CR rates reached 100% for EBV infections and 78.6% for CMV infections. Regarding prophylactic efficacy, no CMV infections occurred in EBV-infected patients after bispecific CTLs infusion, and similarly, no EBV infections developed in CMV-infected recipients following treatment. In terms of safety, no acute or long-term adverse effects were observed. Regarding survival outcomes, with a median follow-up of 175 days (range: 5-1725 days), the 2-year OS was 61.8%. Conclision: Bispecific EBV/CMV-CTLs are a safe and effective therapy for EBV/CMV infections post-allo-HSCT, providing dual-virus coverage for co-infections and prophylaxis against reactivation.