ISCOMATRIX Adjuvant Induces Efficient Cross-Presentation of Tumor Antigen by Dendritic Cells via Rapid Cytosolic Antigen Delivery and Processing via Tripeptidyl Peptidase II

Abstract

D endritic cells (DC)3 are highly specialized APCs that take up exogenous material from the extracellular environment for presentation in the context of MHC molecules. Next to presentation of antigenic epitopes on MHC class II molecules to CD4 ϩ T cells, DCs can also shuttle Ag to the MHC class I processing pathway for CD8 ϩ T cell activation, a process termed crosspresentation (reviewed in Ref. 1). This enables DCs that have engulfed tumor Ag to activate Ag-specific CD8 ϩ T cells capable of tumor cell killing. The cytotoxic antitumor effect of CD8 ϩ T cells also depends on CD4 ϩ T cells, which provide CD8 ϩ T cells with growth factors and costimulatory signals (2-5). Thus, cancer vaccines should aim at inducing both antitumor CD4 ϩ and CD8 ϩ T cell responses.Tumor vaccines based on full-length recombinant proteins have the potential to generate broad-based CD4 ϩ and CD8 ϩ T cell responses (6). However, using recombinant tumor Ag, we found that cross-presentation of soluble Ag by DCs is highly inefficient, whereas presentation to CD4 ϩ T cells was readily induced (7). Delivering tumor Ag to the MHC class I processing machinery in DCs is thus a challenge in the design of cancer vaccines and requires a better understanding of the mechanisms of cross-presentation.Ag processing via MHC class I and II pathways largely depends on the route of Ag uptake. Receptors facilitating cross-presentation include members of the C-type lectin receptor family, FcR, and integrins (reviewed in Ref. 8). Because most CD8 ϩ T cell epitopes are generated in the cytosol, where cleavage of the Ag by the proteasome and other protease complexes occurs (9), Ag translocation from endocytic compartments into the cytosol is essential for effective cross-presentation. Peptides generated by cytosolic proteases are then transported via TAP into the endoplasmic reticulum where additional trimming may occur (10) to allow loading of optimal length peptides onto nascent MHC class I molecules and subsequent transport of the peptide MHC complexes to the cell surface for presentation to CD8 ϩ T cells. Our group has previously shown that cross-presentation of recombinant NY-ESO-1, a 180-aa protein, which is expressed in a variety of human cancers including melanoma, breast, lung, prostate, and others (11,12), by DCs depends on Ag formulation. Using NY-ESO-1 as a model Ag we found that cross-presentation efficiency was enhanced by targeting the Ag to FcR with ICs (7, 13) or by formulation of the Ag with ISCOMATRIX adjuvant, which is being developed for use in human vaccines (7). ISCOMATRIX adjuvant is based on the immune-stimulating complexes (ISCOM) technology, which combines an efficient Ag delivery system with the immunostimulatory activity of saponin and has been shown to promote humoral and cellular immune responses in a variety of animal models (reviewed in Ref. 14). HLA-A2-restricted cross-presentation of NY-ESO-1/immune complex (IC) was proteasome dependent, and for NY-ESO-1/ISCOMATRIX

Processing and cross-presentation of individual HLA-A, -B, or -C epitopes from NY-ESO-1 or an HLA-A epitope for Melan-A differ according to the mode of antigen delivery

“…4,38,39 We found that proteasome inhibition significantly reduced NY-ESO-1 157-165 / HLA-A2, NY-ESO-1 60-72 /HLA-B7, and NY-ESO-1 92-100 /HLA-Cw3-restricted cross-presentation when NY-ESO-1 was presented as an IC (Figure 7). In contrast, proteasome inhibition had no effect on the generation of either the NY-ESO-1 157-165 /HLA-A2 or NY-ESO-1 92-100 /HLA-Cw3 epitopes but dramatically inhibited NY-ESO-1 60-72 /HLA-B7 epitope generation, either when DCs were exposed to formulated NY-ESO-1/ISCOMATRIX vaccine or when NY-ESO-1 and ISCOMATRIX adjuvant were added separately to the DCs ( Figure 7).…”

“…For NY-ESO-1, this may have been in association with cell surface calreticulin, as previously described, 43 which had been internalized into endosome/phagolysosomal compartments. 38 Similar mechanisms may apply for Melan-A, even though cell surface binding with calreticulin has not been described. As previously shown, the addition of ISCOMATRIX likely facilitates translocation into the cytosol, thereby providing tumor antigen access to the class I MHC pathway.…”

“…4,40,41 ISCOMATRIX adjuvant composes 40nM cage-like particles, which can be mixed with antigen or into which hydrophobic antigens can be physically incorporated ("formulated"). We have previously shown that ISCOMATRIX adjuvant dramatically affects antigen processing and presentation, 4,38 and this study was undertaken to more deeply assess the capacity of ISCOMATRIX adjuvant to facilitate the cross-presentation of multiple tumor antigen-derived CTL epitopes. Comparisons have also been made with antigen delivered as ICs.…”

“…As previously shown, the addition of ISCOMATRIX likely facilitates translocation into the cytosol, thereby providing tumor antigen access to the class I MHC pathway. 38 We next evaluated the effect of DC activation on crosspresentation. Class I MHC antigen processing is largely dependent on the proteasome, which has alternative forms depending on immune activation.…”

“…These studies demonstrated that the HLA-A2-restricted cross-presentation of each of these NY-ESO-1 antigen formulations required endosomal acidification but were either proteasome independent or proteasome dependent, respectively. 4,38 To more fully understand the mechanisms used by DCs for the efficient cross-presentation of NY-ESO-1 and Melan-A, we again inhibited components of the antigen-processing machinery. As shown in Figure 3, inhibition of endosomal acidification via the vacuolar type H[ϩ]-ATPase protein pump inhibitor, ConB, abolished NY-ESO-1 157-165 /HLA-A2-restricted cross-presentation of the NY-ESO-1/ISCOMATRIX vaccine as well as inhibited both the crosspresentation of NY-ESO-1 157-165 /HLA-A2 and Melan-A 26-35 /HLA-A2 epitopes when antigen and adjuvant were added separately to the MoDC cultures.…”