Inducible MyD88/CD40 (iMC) Costimulation Enhances Polyclonal Epstein-Barr Virus-Specific Cytotoxic T Lymphocyte (EBV-CTL) Proliferation and Anti-Tumor Activity

Introduction: Adoptive T cell transfer with antigen-specific cytotoxic T lymphocytes (CTLs) have shown promise for the treatment of cancer. Antitumor efficacy correlates with T cell expansion and persistence following infusion, suggesting that enhancing T cell proliferation and survival may increase the potency of T cell therapies. Here, we demonstrate that genetic modification of EBV-specific T cells with inducible MyD88/CD40 (iMC) provides user-controlled costimulation that amplifies IL-2 production, CTL proliferation and cytotoxicity against EBV-transformed B cells, suggesting that iMC may be used to augment EBV-CTL against EBV-associated malignancies.

Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were used to generate EBV lymphoblastoid cells (LCLs) by transforming B cells with B95-8 virus. EBV-CTL were subsequently generated by stimulating PBMCs with irradiated (40 Gy) autologous LCLs at a T cell to LCL ratio of 40:1 (day 0) and then subsequently expanded for an additional 2 weeks by restimulation (4:1 CTL:LCL ratio) in the presence of 50 U/ml IL-2. EBV-CTL were then transduced with bicistronic retroviral vectors encoding iMC and truncated CD19 (ΔCD19) (SFG-iMC-ΔCD19) or a control vector lacking the MC costimulatory domains (SFG-FKBP-ΔCD19). EBV specificity and transduction efficiency of EBV-CTL was assessed by EBV-specific dextramers and CD3⁺CD19⁺ expression by flow cytometry, respectively. In vitro antitumor activity and T cell proliferation was measured using a 7 day live cell imaging (IncuCyte) coculture assays with transduced EBV-CTL and autologous or HLA-disparate LCL with or without 1 nM rimiducid to activate the iMC costimulatory molecule at effector to target (E:T) ratios of 40:1, 20:1, and 10:1. Cytokine production was measured by ELISA.

Results: EBV-CTL were successfully generated from EBV-seropositive donors and demonstrated specificity against EBV lytic antigens (EBNA3A; BZLF1) and showed specific recognition of autologous EBV-LCL, but not HLA-mismatched LCL. EBV-CTL were also efficiently transduced with iMC and control (FKBP) retroviral vectors (93% and 82%, respectively). Cytokine production (IL-2 and IL-6) by gene-modified EBV-CTL targeting autologous LCL was significantly enhanced by iMC activation with rimiducid (48-fold increase compared to FKBP-ΔCD19-transduced EBV-CTL and iMC-ΔCD19-transduced EBV-CTLs without rimiducid stimulation). In vitro live cell imaging demonstrated at high E:T ratios (40:1) that EBV-CTLs efficiently killed matched LCLs. However, at lower E:T ratios (20:1 and 10:1), only EBV-CTLs transduced with iMC-ΔCD19 and stimulated with rimiducid were able to control LCL outgrowth over 7 days, showing a 10-fold increase inhibition in tumor growth.

Conclusion: iMC provides a flexible, costimulatory module that can be used to enhance EBV-specific CTL to increase anti-tumor activity. Activation of iMC with rimiducid, in combination with recognition of cognate EBV antigens, induces IL-2 production and promotes CTL proliferation and survival. These data suggest that genetic modification of EBV-CTL, or other virus or tumor-associate antigen-specific T cells, including tumor infiltrating lymphocytes (TILs), with iMC may provide necessary costimulation on demand to increase T cell proliferation and persistence leading to increased anti-tumor efficacy.