Abstract B18: Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI)

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 a