Tumor-Derived Autophagosome Vaccine: Mechanism of Cross-Presentation and Therapeutic Efficacy

Abstract

Purpose We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8+ T cells. Here we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), derived from tumor cells after inhibition of protein degradation and provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy. Experimental Design DRibbles were characterized by western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed and the anti-tumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma. Results The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiPs), as well as damage-associated molecular pattern (DAMP) molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) and cross-presentation was partially CLEC9A-dependent. Furthermore, this autophagy assisted antigen cross presentation pathway involved both caveolae- and clathrin-mediated endocytosis and ERAD machinery. It depends on proteasome and TAP1, but lysosome functions of APCs. Importantly, DC loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma. Conclusion These data documented the unique characteristics and potent anti-tumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.

Sorafenib and a novel immune therapy in lung metastasis from hepatocellular carcinoma following hepatectomy: A case report

“…Our studies demonstrated that antigens sequestered in the autophagosome may be delivered to DCs for cross-presentation and prime naïve antigen-specific CD8 T cells effectively (17). Subsequently, autophagosomes containing a broad spectrum of cellular antigens from antigen donor cells were collected by induction of autophagy and inhibition of lysosomal̸proteosomal activity, and named DRibbles (9,14,18,19). DRibbles have been demonstrated to exhibit vigorous antitumor efficacy in mouse experiments and cross-prime human CD8 T cells in ex vivo studies (10,11).…”

“…DRibbles was found to express abundant CLEC9A ligand and may directly bind to CLEC9A expressed on the surface of CD141 + cDC subsets. This suggests that the CD141 + cDC subset may be involved in the recognition or subsequent procession of DRibbles by DCs (14). In the presence of GM-CSF and IFN-α, monocytes may differentiate into IFN-DCs in vitro, which may have a more potent function in terms of CD8 T-cell cross-priming compared with traditional interleukin (IL)-4-DCs generated from monocytes stimulated by GM-CSF and IL-4 (13,24).…”

“…IFN-DCs were administered by subcutaneous injection (10 7 cells each time) on days 4, 18 and 31. HCC cell line SMMC-7721-derived DRibbles was prepared as previously described (14). Briefly, SMMC-7721 cells were treated with bortezomib (200 nmol̸l) and ammonium chloride (10-20 mmol̸l) for 24-48 h. DRibbles was dislodged from cells or large cell debris by rigorous pipetting.…”

In situloading of skin dendritic cells with apoptotic bleb-derived antigens for the induction of tumor-directed immunity

“…An alternative mechanism underlying the increased cross-presentation potential of blebs is the presence of defective ribosomal products, which are present in the endoplasmic reticulum (an organelle actively translocated into blebs 20 ), potential TAAs that have been described to be efficiently cross-presented. 37,38 However, this concept is speculation, as we currently have no data to support this hypothesis. In any case, bleb-derived antigen is clearly efficiently ingested and cross-presented by skin DC and direct i.d.…”

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer

“…This results in accumulation of proteins including SLiPs and DRiPs in stable double-membrane autophagosomal vesicles that are isolated through cell disruption and differential centrifugation. 21-24 A human allogeneic autophagosome vaccine, DPV-001, comprising DRibbles derived from an adenocarcinoma (UbiLT6) and a mixed histology adenocarcinoma/squamous cell cancer (UbiLT3) cell line, has recently been tested in a randomized multi-center Phase II study for adjuvant treatment of definitively treated stage IIIA/B NSCLC patients and a pilot study for men with advanced prostate cancer. 24 Gene expression analysis of the two cell lines, UbiLT3 and UbiLT6, revealed that they highly express genes for a wide range of antigens common to adenocarcinomas and squamous cancers (unpublished data).…”