Abstract
SummaryThe cross-presenting capacity of dendritic cells (DCs) can be limited by non-specific degradation during endosome maturation. To bypass this limitation, we present in this study a new Accum-based formulation designed to promote endosome-to-cytosol escape. Treatment of primary DCs with Accum linked to the xenoantigen ovalbumin (OVA) triggers endosomal damages and enhances protein processing. Despite multiple challenges using ascending doses of tumor cells, DC prophylactic vaccination results in complete protection due to increased levels of effector CD4 and CD8 T cells as well as high production of pro-inflammatory mediators. When combined with anti-PD-1, therapeutic vaccination using both syngeneic and allogeneic Accum-OVA-pulsed DCs triggers potent anti-tumoral responses. The net outcome culminates in increased CD11c, CD8, and NK infiltration along with a high CD8/Treg ratio. These highly favorable therapeutic effects highlight the promising potential of Accum as a distinct and potent technology platform suitable for the design of next generation cell cancer vaccines.
Highlights
Anti-tumoral immunity relies on antigen cross-presentation by dendritic cells (DCs).[1,2] For this process to occur, soluble antigens must be first engulfed and sorted into endosomes, whose main function is to initiate limited degradation of captured antigens by lysosomal proteases so they can be exported to the cytosol as large polypeptide fragments for further processing by the proteasomal complex.[3]
Biochemical characterization of aOVA bioconjugate To generate the aOVA final product, a chemical reaction linking an Accum moiety to lysine residues of nOVA was performed (Figure 1A). This led to changes in the molecular weight of the protein, as shown by a smear detected by Coomassie staining (Figure 1B, left) and western blot (Figure 1B, right)
Since chemical modifications of proteins can affect their physio-chemical properties, we assessed the overall stability of aOVA by measuring protein unfolding following thermal stress
Summary
Anti-tumoral immunity relies on antigen cross-presentation by dendritic cells (DCs).[1,2] For this process to occur, soluble antigens must be first engulfed and sorted into endosomes, whose main function is to initiate limited degradation of captured antigens by lysosomal proteases so they can be exported to the cytosol as large polypeptide fragments for further processing by the proteasomal complex.[3] The generated eight to nine amino acid peptides are loaded onto cell surface major histocompatibility complex (MHC) class I molecules to activate responding CD8 T cells.[3] Since the proteolytic activity of endosomal proteases is optimal at acidic pH, maturing endosomes undergo progressive acidification through the recruitment of several V-ATPase subunits.[4,5,6,7,8,9] Interestingly, some DC subsets such as CD8+DCs in mice (described as the equivalent to CD141+XCR1+ in humans10), have developed specific means to minimize endocytic acidification in order to protect captured antigens from uncontrolled or exacerbated degradation.[8] One of these mechanisms consists of assembling the NADPH oxidase (NOX)[2] in CD8+DC endosomes, which would lead to reactive oxygen species production within endosomal lumen to prevent acidification.[4,5,6,7,8,9] This explains the distinct cross-presentation abilities of CD8+DCs compared with monocyte-derived CD8ÀDCs.[8] the use of these cross-presenting DCs for the development of an ex vivo DC vaccine pulsed with an antigenic preparation is difficult to achieve with their limited number in peripheral blood of mice and humans.[11] Besides, the alternative preparation of cross-presenting DCs for vaccine applications using the induced pluripotent stem technology is both costly and time-consuming.[11] novel strategies must be designed to tightly control or modulate endosomal degradation in monocyte-derived DCs as a means to avoid damaging/destroying antigen fragments important for the generation of immunogenic peptides endowed with the capacity to elicit effective anti-tumoral immunity
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