Anti-CD83 (full length) polyclonal antibody

Dendritic cell (DC)-based antigen-targeted immunotherapy may offer effective adjuvant therapy for hepatocellular carcinoma (HCC), in which cytotoxic T lymphocytes (CTLs) are key. However, in a number of cases, the activity of CTLs is completely inhibited due to the downregulated expression of major human leukocyte antigen (HLA) class I molecules by HCC cells. The aim of the present study was to overcome this issue. Hep3B cells were transduced by HCC-specific recombinant adeno-associated virus (rAAV) carrying human alpha-fetoprotein promoter (AFPp) and the interferon-gamma (IFN-gamma) gene (rAAV/AFPp-IFN-gamma). rAAV carrying the cytomegalovirus promoter (CMVp) and human alpha-fetoprotein (AFP) gene (rAAV/CMVp-AFP) was used to transduce professional antigen-presenting DCs for the purpose of stimulating a CTL response. It was observed that transduction of DCs with rAAV/CMVp-AFP resulted in: (i) AFP and interleukin-12 expression; (ii) high expression levels of cluster of differentiation (CD) 80, CD83, CD86, CD40, HLA-death receptor and CD1a; (iii) T cell populations with marked IFN-gamma expression; (iv) a high percentage of CD69+/CD8+ T cells; and (v) the activity of CTLs against HLA-A2-expressing Hep3B cells. The transduction of Hep3B cells with rAAV/AFPp-IFN-gamma resulted in: (i) IFN-gamma expression; (ii) upregulated expression of HLA-A2; and (iii) an improved CTL response against HLA-A2-deficient Hep3B cells. rAAV/CMVp-AFP-transduced DCs elicited an AFP-specific and HLA-class I-restricted CTL response against Hep3B cells. In conclusion, it was shown that the transduction of Hep3B with rAAV/AFPp-IFN-gamma upregulated the expression of HLA-A2 and improved the sensitivity to CTL response.

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response. This work focuses on the development of predictive in vitro assays that can monitor DC maturation, in order to determine whether drug products have direct DC stimulatory capabilities. To this end, four independent laboratories aligned a common protocol to differentiate human monocyte-derived DC (moDC) that were treated with either native or aggregated preparations of infliximab, natalizumab, adalimumab, or rituximab. These drug products were subjected to different forms of physical stress, heat and shear, resulting in aggregation and the formation of subvisible particles. Each partner developed and optimized assays to monitor diverse end-points of moDC maturation: measuring the upregulation of DC activation markers via flow cytometry, analyzing cytokine, and chemokine production via mRNA and protein quantification and identifying cell signaling pathways via quantification of protein phosphorylation. These study results indicated that infliximab, with the highest propensity to form aggregates when heat-stressed, induced a marked activation of moDC as measured by an increase in CD83 and CD86 surface expression, IL-1 beta, IL-6, IL-8, IL-12, TNF alpha, CCL3, and CCL4 transcript upregulation and release of respective proteins, and phosphorylation of the intracellular signaling proteins Syk, ERK1/2, and Akt. In contrast, natalizumab, which does not aggregate under these stress conditions, induced no DC activation in any assay system, whereas adalimumab or rituximab aggregates induced only slight parameter variation. Importantly, the data generated in the different assay systems by each partner site correlated and supported the use of these assays to monitor drug-intrinsic propensities to drive maturation of DC. This moDC assay is also a valuable tool as an in vitro model to assess the intracellular mechanisms that drive DC activation by aggregated therapeutic proteins.