Abstract 2362: Optimizing prostate cancer immunotherapy by reducing vaccine induced PD-1 expression
Authors: Zahm Christopher D. , McNeel Douglas G. 
Published: July 2016

Abstract
Through early phase clinical trials we have shown that a plasmid DNA vaccine can elicit antigen-specific CD8 T-cells that can persist for years. However, the persistence of antigen-expressing prostate tumors demonstrates that mechanisms of tumor escape are at play. To investigate these mechanisms we have focused on vaccine strategies using the synovial sarcoma, X breakpoint 2 (SSX2) protein as a model target antigen and utilized mice which express HLA-A2 but not murine MHC-I. We have shown that immunization with a DNA plasmid encoding SSX2 elicits robust CD8+ cells and that altering HLA-A2 affinity can augment this response. High affinity altered peptide ligand (APL)-encoding DNA vaccines elicited a greater frequency of specific T-cells; however, they fared worse in anti-tumor studies due to increased PD-1 expression on T-cells, an effect which could be abrogated with PD-1 blockade. Our current research has focused on why high-affinity epitopes elicit higher PD-1 expression, and whether this might be modulated at the time of antigen presentation. To that aim we have used the OT-1 mouse model, in which T-cells express a T-cell receptor that is specific for the dominant ovalbumin epitope SIINFEKL. We generated APLs of SIINFEKL and used them to stimulate OT-1 splenocytes ex vivo. The high affinity SIINFEKL epitope resulted in high, sustained expression of PD-1 following activation, while lower affinity APLs resulted in less, transient expression. However, lowered PD-1 expression can be indicative of T-cells that receive weaker signals via the T-cell receptor (TCR) and likewise display lowered effector function, therefore we also analyzed cytokine production and the in vivo effects of the APLs. In immunization experiments using B6 mice carrying adoptively transferred OT-1 T-cells the lower affinity peptide again resulted in transient PD-1 expression while the high affinity peptide led to sustained levels of PD-1 expression. One week after vaccination with the low affinity APL, PD-1 on tetramer+ CD8 T-cells had returned to baseline while cells from mice immunized with SIINFEKL maintained PD-1 expression. Furthermore, 38(±8)% of tetramer+ cells from the SIINFEKL immunized group were PD-1+ 4-1BB- indicating that PD-1 expression is maintained even after activation in vivo. We are currently assessing the ability of immunization with each peptide to prevent B16-OVA tumor growth in vivo. To assess the mechanism by which PD-1 is regulated after stimulation with each peptide we used a combination of live cell imaging and RNA sequencing to observe APC/T-cell interactions and T-cell gene expression respectively. This research indicates that while increasing antigen affinity may generate a more robust T-cell response it does not always result in better anti-tumor immunity and provides a model in which we can study the biology underlying PD-1 expression.
Citation Format: Christopher D. Zahm, Douglas G. McNeel. Optimizing prostate cancer immunotherapy by reducing vaccine induced PD-1 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2362.