Abstract 2565: EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system
Authors: Tiwari Aradhana Awasthi , McCormick Alison , Edani Dina , Newman Aaron , Azmy Christeen , Ayello Janet , Klein Christian , Cairo Mitchell S. 
Published: August 2018

Abstract
Background: EBV latent proteins (LMP-1/2) increase the chemoresistance in Burkitt Lymphoma (BL) cells by constitutively activating the CD40 receptor. It has also been demonstrated that B- cell lymphoma resistant to rituximab is a result of LMP-1 induced miR-155 expression and survival mediated by AKT phosphorylation (Hong et al.Gene Ther, 2012). EBV peptides delivered by a virus carrier (Tobacco mosaic virus;TMV) has the potential to boost immunity and stimulate potent T cell responses against EBV-associated antigens and induce a virus specific T cell response against EBV infected malignant cells (Miles/Cairo et al. BJH,2012, Kemnade/McCormick et al. Vaccine, 2014).We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al. BJH, 2015) in xenografted NSG mice. However, synergistic effects of obinutuzumab in-combination with LMP1/2-peptide vaccine against mature-B-NHL are unknown.
Objective: To determine the efficacy of peptide specific cytotoxic T-lymphocytes (CTL) or the EBV vaccine alone/ in-combination with obinutuzumab against mature-B-NHL
Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji cells (ATCC, USA) were cultured in 10% RPMI. Tumor cells were incubated with TMV-RIED IgG (LMP2) with obinutuzumab (100ug/ml) for 4 hrs with NK cells. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E:T ratio. Further, to generate LMP1/LMP2 peptide specific cytotoxic T cells (CTL), mature dendritic cells were pulsated with TMV-conjugated LMP1/LMP2 peptides for 24 hrs. PBMC isolated T cells were mixed with APC (LMPs-mature -DCs) at 1:20 ratio for activation and maturation. LMP-peptide specific CTLs were investigated in-vitro for cytotoxicity efficacy against BL cell lines.
Results: LMP1 and LMP2 peptide were successfully conjugated with TMV. TMV conjugated and purified serum vaccine was tested against BL cell lines. Conjugated serum vaccine (LMP2-(RIED-TMV) +obinutuzumab+NK, compared to obinutuzumab+NK or vaccine+NK alone significantly enhanced in-vitro cytotoxicity 62.67±7.82% vs. 53.66±6.43% vs. 50.8±5.2% (p=0.04 and 0.02), respectively against BL (Raji). Furthermore, cytotoxicity of CTL (LMP1-MSD-TMV) or (LMP2-TYG-TMV) cells were significantly enhanced compare to IL2 treated T cells only, 58.5±8.6% or 48.56±15.18% vs. 21.42±13.3% in Raji (P=0.01, 0.02), and 57.8±7.9% or 45.52±19.4% vs. 14.48±4.2% in Raji4RH (P=0.003, 0.002), respectively.
Conclusion: Our preliminary data indicates that LMP1 (MSD-TMV) and LMP2 (TYG-TMV) successfully conjugated with TMV and TMV-conjugated peptide specific CTL cells significantly enhanced cytotoxicity against BL cell lines compared to T cells only.
Citation Format: Aradhana Awasthi Tiwari, Alison McCormick, Dina Edani, Aaron Newman, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. EBV peptide-derived vaccine significantly enhanced in vitro cytotoxicity against EBV-positive B-cell lymphoma (EBV-BL) treatment using TMV-based delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2565.