Background Epstein Barr virus (EBV) is a highly prevalent herpesvirus worldwide and its infection is associated with malignancies such as nasopharyngeal cancer (NPC), gastric cancer, lymphoepithelioma cancer and lymphoproliferative disorders. Virus-specific T (VST) cell therapies using T cells reactive against EBV have shown limited efficacy in clinical trials with several practical limitations such as the long preparation period and the variations of pre-existing EBV-reactive T cells in different donors. To overcome these technical constraints of the VST approach, we have established the genetically engineered T cells targeting EBV antigens with lentiviral transduction. A proof-of-concept clinical validation was further conducted against EBV⁺ malignancies (trial No. ChiCTR2100044497).

Methods T cells were activated by CD3/CD28 stimulation, lentivirally transduced with codon-optimized CAR (targeting EBV envelope protein) or TCR (targeting EBV latent membrane protein) and further expanded ex vivo. Transgene expression and the T cell phenotype were assessed by flow cytometry. In vitro anti-tumor efficacy was analyzed by IFN-γ ELISA and killing assays such as RTCA Xcelligence. In vivo, the antitumor efficacy of EBV-specific T cells was evaluated in immunocompromised mice bearing human tumor xenografts. Anti-tumor responses in lymphoma and NPC were evaluated by Lugano 2014 and RECIST 1.1 respectively.

Results T cells lentivirally transduced with EBV antigen-specific CAR or TCR demonstrated satisfactory surface expression of the introduced receptor and showed increased cytokine production when stimulated to target expressing tumor cells in vitro (figure 1). We have identified the TCRs against EBV latent membrane protein with high functional avidity as determined by peptide dose-response functional assays. In vitro cytotoxicity assays demonstrated strong killing of antigen positive tumor cell lines of the engineered EBV-specific CAR-T or TCR-T cells, while sparing the antigen-negative cells, indicating the specific T cell response. The single intravenous infusion of engineered EBV-specific T cells also led to dose-dependent anti-tumor responses in immunocompromised mouse xenograft models (figure 1). In the clinical trial, two patients with lymphoproliferative disease and one patient with advanced NPC treated with engineered EBV-targeting T cells experienced sustained objective responses. Moreover, in the NPC patient, the EBV DNA copy number decreased from 3.32*10³ copies/ml at baseline to normal (<5.00*10² copies/ml) 2 months post infusion. The infused cells also demonstrated satisfactory safety profile with no neurotoxicity and ≥ Grade 2 cytokine release syndrome observed.

Conclusions Our data demonstrate the feasibility of using genetically engineered T cells targeting EBV antigens such as envelope protein or latent membrane protein to treat EBV-associated malignancies.

Trial Registration Chinese Clinical Trial Registry: ChiCTR2100044497

Ethics Approval All animal studies were conducted at Guangzhou Regenerative Medicine and Health, Guangdong Laboratory as approved by IACUC (2020125). The clinical study was approved by the first affiliated hospital of Zhengzhou University’s Ethics Board (L2021-Y027–005).

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Abstract 328 Figure 1

Epstein Barr Virus (EBV)-specific CAR-T or TCR-T against EBV-associated malignancies. (A) Schematic overview of the EBV specific CAR or TCR construct. SR signal peptide. TM, transmembrane domain. ICD, intracellular domain. (B) Flow cytometric analysis of the CAR or TCR expression on T cell surface. (C) EBV antigen expression on clinical tumor samples by immunohistochemistry analysis. Top, envelope glycoprotein expression on EBV+ lymphoma tissues; Bottom: latent membrane protein expression on nasopharyngeal cancer tissues. Scale bar, 50 µm. (D) In vitro functional evaluation of EBV-specific CAR-T. CAR-T were co-cultured with lymphoblastoid cell line (LCL) with indicated E:T ratio and supernatant was subjected to IFN-gamma ELISA and LDH-based cytotoxicity assay (performed after 16 h of co- culture). (E) Mouse survival curve. Jurkat cells expressing EBV envelope protein (Jurkat-Luc/ZT009) was inoculated into the tail vein of NSG mice and five days later effector cells were injected i.v. on Day 0. The dose Of the CAR-T was set three doses, 5x106, 1x107 and 2x107 respectively. The survival was monitored until the moribund of all mice from control group. (F) In vitro functional evaluation of EBV-specific TCR-T. TCR-T were co-cultured with C666.1 with indicated E:T ratio and supernatant was subjected to IFN-gamma ELISA and LDH-based cytotoxicity assay (performed after 16 h of co-culture). (G) In vivo testing of TCR-T cells in B- NDG mice xenografted with HLA-matched C666.1 cell line expressing latent membrane protein (5x106 cells/s.c.). Tumor volume was measured till Day 30 post T cell inoculation. (H) The pharmacokinetics analyses of the three patients as determined by copies/ml whole blood by qPCR using primers specific to the CAR/TCR construct.