Ubiquitinated Proteins Collected By Ubiquitin Binding Adaptor Protein-Vx3 As A Novel Cancer Vaccine

Background:Previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles:DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, it was sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine.Methods:1-The lysates from two tumor cell lines, EL4 and B16-F10, were prepared after their proteasomal degradation pathway blocking by velcade and NH4C1 followed by enrichment of ubiquitinated proteins using two different ubiquitin binding proteins, tandem ubiquitin binding entities (TUBEs) and Vx3(A7) protein. The isolated ubiquitinated proteins were detected by SDS-PAGE analysis and western blotting technique.2-The specific anti-tumor immune response induced by ubiquitinated proteins was detected by vaccination C57BL/6 mice with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA and intracellular staining. 3-Two tumor models, EL4 and B16-F10 were established in C57BL/6 mice. Mice were vaccinated with Ub-enriched proteins and PBS, Vx3(A7) protein, whole cell lysate and Ub-depleted proteins (as controls) via inguinal lymph nodes or subcutaneous injection to detect the anti-tumor efficacy of ubiquitinated proteins in the homologous and non-homologous tumor mice model. The tumor growth in the established tumor mice models was monitored.Results:1-Results showed that much more ubiquitinated proteins could be enriched from tumor lysates by Vx3(A7) protein compared with TUBEs. Furthermore, The K63 protein was effectively isolated from tumor lysates using the Vx3(A7) protein. Based on these findings, the Vx3(A7) protein was used as a tool to purify ubiquitinated proteins which then tested whether they able to induce a specific anti-tumor immune response and anti-tumor efficacy in the established tumor models.2-Results revealed that, after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN^y in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-y level than that via subcutaneous injection. Moreover, the level of secreted IFN-y in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-y when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Results also revealed that both Ub-enriched proteins and DRibbles could induce a strong tumor specific immune response without an obvious difference.3-Moreover, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups.Conclusions:1-Vx3(A7) protein is more efficient than TUBEs to enrich the ubiquitinated proteins from tumor cell lysate after their proteasomal degradation pathway inhibition.2-Ub-enriched proteins could induce a strong tumor specific immune response compared to tumor lyaste and Ub-depleted proteins.3-Ub-enriched proteins have a potent anti-tumor efficacy on homologous tumor and expand a cross protection on allogeneic tumor.