Anti-Tumor Efficacy of an Adjuvant Built-In Nanovaccine Based on Ubiquitinated Proteins from Tumor Cells.

BACKGROUND AND AIM: We have previously identified ubiquitinated proteins (UPs) from tumor cell lysates as a promising vaccine for cancer immunotherapy in different mouse tumor models. In this study, we aimed at developing a highly efficient therapeutic adjuvant built-in nanovaccine (α-Al2O3-UPs) by a simple method, in which UPs from tumor cells could be efficiently and conveniently enriched by α-Al2O3 nanoparticles covalently coupled with Vx3 proteins (α-Al2O3-CONH-Vx3). METHODS: The α-Al2O3 nanoparticles were modified with 4-hydroxybenzoic acid followed by coupling with ubiquitin-binding protein Vx3. It was then used to enrich UPs from 4T1 cell lysate. The stability and the efficiency for the UPs enrichment of α-Al2O3-CONH-Vx3 were examined. The ability of α-Al2O3-UPs to activate DCs was examined in vitro subsequently. The splenocytes from the vaccinated mice were re-stimulated with inactivated tumor cells, and the IFN-γ secretion was detected by ELISA and flow cytometry. Moreover, the therapeutic efficacy of α-Al2O3-UPs, alone and in combination with chemotherapy, was examined in 4T1 tumor-bearing mice. RESULTS: Our results showed that α-Al2O3-UPs were successfully synthesized and abundant UPs from tumor cell lysate were enriched by the new method. In vitro study showed that compared to the physical mixture of α-Al2O3 nanoparticles and UPs (α-Al2O3+UPs), α-Al2O3-UPs stimulation resulted in higher upregulations of CD80, CD86, MHC class I, and MHC class II on DCs, indicating the higher ability of DC activation. Moreover, α-Al2O3-UPs elicited a more effective immune response in mice, demonstrated by higher IFN-γ secretion than α-Al2O3+UPs. Furthermore, α-Al2O3-UPs also exhibited a more potent effect on tumor growth inhibition and survival prolongation in 4T1 tumor-bearing mice. Notably, when in combination with low dose chemotherapy, the anti-tumor effect was further enhanced, rather than using α-Al2O3-UPs alone. CONCLUSION: This study presents an adjuvant built-in nanovaccine generated by a new simple method that can be potentially applied to cancer immunotherapy and lays the experimental foundation for future clinical application.

Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy.

A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al2O3 nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al2O3-Vx3) via a chemical linker. The functionalized α-Al2O3-Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al2O3-Vx3-UPs than the physical mixture of α-Al2O3 and Vx3-UPs (α-Al2O3/Vx3-UPs). Laser confocal microscopy images revealed that α-Al2O3-Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al2O3-Vx3-UPs to CD8+ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al2O3-Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al2O3/cell lysate or α-Al2O3/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al2O3 as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines.

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.