Abstract B18: Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI).
Authors: Chinn Jason , Cummings Crystal , Wagener Felecia , Sridhar Shaarwari , Khuu-Duong Kien , Ge Xinhui , Yoshino Karen , Li Sam , Martel Lisa , Ramsborg Chris , Brasel Ken , Trager James , Meagher Craig 
Published: August 2014

Abstract
Sipuleucel-T (Provenge®), indicated for the treatment of asymptomatic or minimally symptomatic metastatic castration resistant prostate cancer, is the first FDA-approved Active Cellular Immunotherapy (ACI). Here we describe the development of a therapeutic ACI for the treatment of renal, lung, colon, and cervical cancer. Like PROVENGE®, this new ACI utilizes a recombinant antigen consisting of carbonic anhydrase IX (CA9) linked to GM-CSF (CA9:GM-CSF). In these studies, we investigated the effects of incorporating selective TLR9 agonists on in vitro measures of ACI potency.
Initially, employing a panel of proprietary TLR9 agonists, we compared the phenotype of antigen presenting cells (APC) following culture of PBMC from normal healthy donors with either CA9:GM-CSF or CA9:GM-CSF plus TLR9 agonist. While antigen derived GM-CSF activity matured APC, characterized by elevated cell surface expression of CD40, CD54, CD80 and CD86, proprietary agonists for TLR9 further enhanced expression of CD40, CD80, and CD86. By extension, stimulation via TLR9 elicited increased costimulatory capacity of CD14+ large APC in allogeneic mixed lymphocyte response assays. Elevated levels of MCP1-3 in culture supernatant were consistent with APC activation, and notably, viability of CD14+ large APC was unaffected following TLR9 stimulation within the ACI product. Interestingly, using an HLA class II restricted CA9-specific T cell hybridoma reporter assay, staggering the addition of CA9:GM-CSF prior to TLR9 agonist was important to maximize antigen uptake and peptide presentation. Collectively, antigen derived GM-CSF activity is robust at activating APC and certain TLR9 agonists strengthen this effect.
Next, as determined by cell surface expression of activation markers (CD27, CD38, CD40, CD54, CD86, IgD), the potential for TLR9 agonists to activate B cells was examined. Relative to cultures supplemented with only CA9:GM-CSF, TLR9 agonists were sufficient to produce a generalized B cell activation pattern consisting of enhanced cell surface expression of CD38, CD40, CD86, and CD54; while expression of CD27 was decreased. Expression of IgD remained unchanged. Associated with the prominent pattern of B cell activation was a marked increase in the accumulation of proinflammatory Type-1 like growth factors (IFN-α, IFN-γ, CXCL9, CXCL10, CCL3, and CCL4) in response to each TLR9 agonist. Despite the degree of B cell activation, this phenotype did not correlate with cellular activation of any T cell subset; suggesting the inflammatory signature resulting from TLR9 stimulation is primarily driven by B cells and/or APC in the ACI setting. Importantly, IL-10 levels were significantly enhanced following TLR9 stimulation, raising the possibility for enhanced regulatory cell activity, but changes in the frequency of regulatory CD4+ T cells were not observed. However, continuing studies are aimed at examining whether regulatory B cell numbers may be enhanced. Thus, in the ACI product, TLR9 agonists potentiate B cell activation and represent powerful polarizers of predominantly Type 1-like cytokine responses.
Overall, agonists of TLR9 are compatible with the ACI platform for potentially enhancing antigen specific immunotherapy of cancer. In current experiments, the capacity of TLR9 agonists to enhance cross-priming and generate cytotoxic T cell responses is under investigation.
Citation Format: Jason Chinn, Crystal Cummings, Felecia Wagener, Shaarwari Sridhar, Kien Khuu-Duong, Xinhui Ge, Karen Yoshino, Sam Li, Lisa Martel, Chris Ramsborg, Ken Brasel, James Trager, Craig Meagher. Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI). [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 B18.