Program: Oral and Poster Abstracts
Session: 801. Gene Therapy and Transfer: Poster II
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.
Disclosures: Khalil: Bellicum Pharmaceuticals: Employment. Yvon: Bellicum Pharmaceuticals: Equity Ownership. Mahendravada: Bellicum Pharmaceuticals: Employment. Spencer: Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster: Bellicum Pharmaceuticals: Employment, Other: stockholders .
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