EBV-Specific CD19 Chimeric Antigen Receptor T Cells Display Robust Antitumor Efficacy Against EBV-Associated B-Cell Malignancies

Background

The treatment of Epstein-Barr virus (EBV) associated B-cell malignancies are still mainly based on chemotherapy with poor outcomes. Therefore, novel approaches for treating EBV-associated B cell malignancies are urgently needed. Chimeric Antigen Receptor T Cells (CAR-T) therapy has achieved great success in B-cell malignancies, while virus-specific T cells (VSTs) have effectively treated virus-associated diseases and malignancies after hematopoietic stem cell transplant (HSCT). Hence, we engineered EBV-CTL with anti-CD19 CAR (EBV/CD19 CAR-T ) and evaluated its cytotoxicity in vivo and in vitro.

Methods

Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to generate EBV-transformed lymphoblastoid cell lines (LCL). EBV/CD19 CAR-T was expanded by established EBV-CTL stimulated with autologous LCL. CD19 CAR-T cells stimulated with CD3/CD28 Dynabeads were applied as a baseline comparison. The co-culture assay was used to evaluate the cytotoxicity of CAR-T cells. The viral load of EBV were detected by qPCR, and the secretion of specific immune cytokines was determined by multi-cytokine assay. NSG mice received an intravenous injection of 5E5 fluorescence-firefly-luciferase tagged Farage (Farage-FFluc). On day 7, mice were injected with a single intravenous dose of normalized EBV/CD19 CAR-T, CD19 CAR-T, EBV-CTL, mock T-cells, and PBS. Tumor burden was monitored once weekly by with IVIS imaging system. EBV load was measured by qPCR. Overall survival was analyzed using the Kaplan-Meier log-rank test.

Results

During 12 days of culture, the expansion kinetics and CD19 CAR transduction efficiency of primary human T cells were comparable between EBV/CD19 CAR-T and CD19 CAR-T(116.91±3.37 VS 120.67±2.59), almost the same. Looking at CD8+/ CD4+ ratios by flow cytometry, EBV/CD19 CAR-T trended toward preferentially expanding CD8+ over CD4+ T cells, as compared with CD19 CAR-T(2.71±0.13 VS 0.80±0.03). EBV/CD19 CAR-T cells exhibited a central memory-type phenotype(CD45RA-CD62L+ ), indicating greater proliferative capacity and extended lifespan. Both EBV/CD19 CAR-T and CAR-T cells expressed low levels of PD-1 and moderate levels of TIM-3, which helps maintain T-cell functionality. To assess whether chronic CAR-mediated activation could lead to T cell dysfunction, we employed a co-culture system whereby per 24 h EBV/CD19 CAR-T or CD19 CAR-T cells were recursively transferred to culture dishes seeded with tumor cells, adjusting for a constant viable E:T ratio=1:2. EBV/CD19 CAR-T cells showed superior antitumor efficacy in five repetitive stimulation rounds of tumor-load-killing assays (EBV/CD19 CAR-T 93.86±0.85% VS CD19 CAR-T 71.93±2.54%, P=0.0012). Cytokine analysis revealed an increased secretion of IL-2, TNF-α, IFN-γ, FasL Granzyme A, and Granzyme B in coculture with EBV/CD19 CAR-T cells compared to CD19 CAR-T cell. Next, we compared the in vivo antitumor efficiency of CD19 CAR-T cells produced using either LCL-based or Dynabeads-based expansion in a xenograft mouse model of EBV-associated B-cell malignancies. In PBS-treated groups, tumor burden progressed rapidly. By day 21 post T cell administration, these mice had died or needed to be euthanized. In contrast, both EBV/CD19 CAR-T and CD19 CAR-T, treated groups cleared the high tumor burdens and were all still alive at the end of the experiment, except for one likely unrelated death. In contrast, mice receiving EBV/CD19 CAR-T were significantly protected from rapid progression (median survival (EBV/CD19 CAR-T VS CD19 CAR-T VS EBV-CTL VS mock T-cells VS PBS): unfollowed VS unfollowed VS 49.5 days VS 43 days VS 29 days). At the time of sacrifice on day 28 post tumor engraftment, EBV DNA load in each organ tissue (bone marrow, spleen, and liver) and peripheral blood from EBV/CD19 CAR-T groups and CD19 CAR-T groups was significantly lower compared to PBS group, especially in the liver (Log₁₀ viral load at liver(EBV/CD19 CAR-T VS CD19 CAR-T VS EBV-CTL VS mock T-cells VS PBS): below the lower limit of detection VS 2.74±0.03 VS 4.35±0.18 VS 5.34±0.10 VS 6.97±0.18 copies/mL) And there are no observed treatment-related toxicities.

Conclusions

Overall, our study indicates that EBV/CD19 CAR-T cell, combining natural receptors for EBV antigens and chimeric receptors for CD19, is a promising strategy in the clinical treatment of EBV-positive B-lymphocyte malignancies.

No relevant conflicts of interest to declare.