Abstract B71: Protective immunizations using cultured peripheral blood cells.
Authors: Brasel Kenneth A. , Meagher Craig , Ligocki Marykay , Cerretti Lauren , Wagener Felecia , Li Sam , Trager James 
Published: August 2014

Abstract
Murine models of Autologous Cellular Immunotherapy (ACI) have been useful tools for understanding the mechanisms by which human immunotherapy may work. Sipuleucel-T is an FDA approved therapeutic for the treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant metastatic prostate cancer; Phase 3 studies demonstrated a statistically significant prolongation in overall survival when compared to a control group. Sipuleucel-T is manufactured by activating peripheral blood mononuclear cells (PBMC), including antigen presenting cells, with a recombinant protein fusion of prostatic acid phosphatase and granulocyte macrophage colony-stimulating factor (GM-CSF). We have previously reported a method for the use of cultured PBMC in a pre-clinical setting to elicit a T cell response against a tumor associated antigen, and to protect mice from tumors expressing that antigen. The model antigen we used was human Carbonic Anhydrase IX (hCA9). Mouse PBMCs were cultured with the antigen fused to murine GM-CSF (hCA9-GM) or with GM-CSF alone. Mice were immunized 3 times with these cells at 2 week intervals. Satellite mice were then harvested for evaluation of in vitro T cell response (antigen recall assay) and the remaining mice were inoculated with hCA9-expressing tumor cells, and tumor growth followed over time. Immunized mice mounted a robust T cell response in vitro to hCA9 in a dose dependent manner, a result that correlated with protection from tumor challenge in mice immunized with PBMC cultured in hCA9-GM.
Herein, we investigate the cellular requirements for protection against tumor challenge and ability of a selective Toll-like receptor (TLR) 9 agonist to enhance the potency of this cellular immunotherapy. To investigate the importance of T cells for anti-tumor responses, we demonstrate that selective depletion of either CD4 or CD8 T cells results in a diminution or loss of protection toward tumor challenge; indicating T cells are necessary in vivo to prevent tumor growth. Next, to stimulate antigen specific T cell activity in the ACI therapy, relative to hCA9 antigen alone, cells were cultured with hCA9-GM antigen in combination with the TLR9 agonist CpG1826. Addition of CpG1826 resulted in significant accumulation of pro-inflammatory cytokines (IL-1b, IL-6, IL-10, TNFa, KC and IFNg) in the culture medium, and to higher surface expression of activation and co-stimulatory markers (CD25, CD69, CD80, CD86, and MHC Class II) on cultured cells. Moreover, inclusion of CpG1826 to cultures enhanced protection against tumor cell challenge. Interestingly, this enhancement in potency was not associated with increased antigen specific T cell responses, as measured in the in vitro antigen recall assay.
In conclusion, we show that, in mice, unfractionated white blood cells collected from peripheral blood can be harnessed to mount effective anti-tumor immune responses and that TLR9 agonists complement this approach. Furthermore, this active immunotherapy triggers a balanced immune response relying on the activity of both CD4 and CD8 T cells. Future studies will be aimed at evaluating the role of various PBMC subsets (T cell, B cell and myeloid cell) in protecting mice from tumor cell challenge.
Citation Format: Kenneth A. Brasel, Craig Meagher, Marykay Ligocki, Lauren Cerretti, Felecia Wagener, Sam Li, James Trager. Protective immunizations using cultured peripheral blood cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B71.