Abstract 1928: Resistance of human melanoma to MART-1/HLA A2.1 specific CTL (F5 CTL)-killing: poor recognition, low MART-1 expression and reversal of resistance

Adoptive cell therapy (ACT) using ex vivo engineered autologous lymphocytes encoding high affinity TCRα/β genes specific for MART-1 + /HLA-A2.1 + (F5 CTL) kill human melanoma lines in vitro in an MHC-I restricted and caspase-dependent manner and mediate modest clinical responses. Development of antigen-loss tumor variants is one mechanism of immune escape. The objective of this study is to investigate the mechanism of resistance to CTL-killing due to loss of recognition and reversal of resistance by pharmacological inhibitors. We hypothesized that the development of Ag-loss variants is due to degradation or epigenetic modifications of MART-1. Also, we hypothesized that aberrant apoptotic cell signaling contribute to immune resistance. To test the hypotheses, we generated F5 CTL-resistant clones (R) from sensitive parental (P) lines by growing the cells under F5 CTL selective pressure followed by limiting dilution. Detailed analysis revealed that compared to P cells, M244R clone exhibit significantly higher resistance to F5 CTL killing, proliferate at a higher rate, and are unable to trigger IFNγ release by F5 CTLs suggesting diminished MART-1 expression (loss of MART-1 was confirmed by gene analysis, surface HLA-A2.1 was unaltered). F5 CTLs recognized and interacted with MART-1 pulsed M244R clone (IFNγ secretion and conjugate formation), yet, the cells still remained resistant to F5 CTL killing. We observed deregulation and constitutive hyper-activation of the components of the NF-κB pathway leading to over-expression of anti-apoptotic Bcl-2, Mcl-1 and Bcl- xL in M244R clones. The proteasome inhibitor bortezomib efficiently blocked NF-κB activity, reduced the expression of resistant factors, restored recognition and sensitized the M244R clones to F5 CTL-induced killing. These novel results suggest that bortezomib, not only interferes with the deregulated signaling pathway but also restores recognition of low MART-1 expressing, poorly recognized immune resistant variants by F5 CTLs, thus, reverses the acquired resistance. These results also imply that immune resistance can not be solely explained by the development of MHC-/antigen-loss variants and is partly due to perturbed cell survival signaling pathways of the tumor cells. Targeted therapy with specific pharmacological inhibitors in conjunction with F5 CTL can override F5 CTL-resistance, which can improve clinical response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1928.