Abstract 3979: In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy.
Authors: Kim Peter , Hodge James , Grenga Italia , Donahue Renee , Schlom Jeffrey , Farsaci Benedetto 
Published: August 2014

Abstract
A previous murine study (B. Farsaci et al, Int. J. Cancer, 2010) showed that mice administered an anti-cancer vaccine prior to treatment with the pan-BCL-2 inhibitor obatoclax (GX15-070) showed a substantial reduction of metastatic mouse lung tumors due to increased apoptotic resistance of mature CD8T cells and decreased Treg function.
The purpose of this study was to evaluate activity of obatotclax on human lymphocyte populations. In vitro effects of obatoclax on human T cell subsets (naïve, central memory, effector-memory and terminal effector T cells) at different maturation states (CD69+ early-activated T cells and CD69- mature T cells) and regulatory T cells (Tregs) were evaluated at multiple concentrations (0.1 - 10 uM). Purified CD8 T cells or whole PBMCs from normal donors were activated for 3-4 days with anti-CD3 and anti-CD28 monoclonal antibodies (short-term culture) or maintained in an IL7/IL15-enriched medium for 7 additional days (long-term culture). After short- or long-term cultures, isolated CD8Ts and whole PBMCs were treated with obatoclax or control (DMSO) for 24 hours, and their viability and differentiation status were analyzed by flow cytometry.
The results indicate that (1) long-term cultured CD8 and CD4T cells were more resistant to obatoclax induced cell death than those in the short-term culture condition, (2) obatoclax increased the percentage of total memory CD8 and CD4T cells (central memory + effector memory) in the long-term culture condition, and (3) obatoclax increased the apoptosis of regulatory T cells (Tregs) and induced a profound down-regulation of FOXP3 in a dose dependent manner.
It is postulated that first, for an optimum combination effect, an immune-stimulating agent directed at T cells needs to precede obatoclax treatment long enough to allow T cells to acquire resistance to obatoclax. Then secondly, with proper treatment scheduling, memory T cells can be viably maintained, thereby establishing a sustainable source of cytotoxic T cells to kill tumors. Finally, because human regulatory T cells are sensitive to obatoclax, there is a potential for obatoclax to tip the numerical and functional balance of effector T cells and Tregs towards effector T cells in human tumor microenvironment. These conclusions support the preclinical murine in vivo study, and provide the rationale for combining obatoclax with an immunotherapeutic regimen in clinical studies.
Citation Format: Peter Kim, James Hodge, Italia Grenga, Renee Donahue, Jeffrey Schlom, Benedetto Farsaci. In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2013-3979