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Fang Huang,1,* Jinjin Zhao,1,* Yiting Wei,1 Zhifa Wen,1 Yue Zhang,1 Xuru Wang,1 Yanfei Shen,2 Li-xin Wang,1 Ning Pan1 1Department of Microbiology and Immunology, Medical School of Southeast University, Nanjing, Jiangsu Province 210009, People’s Republic of China; 2Department of Bioengineering, Medical School of Southeast University, Nanjing, Jiangsu Province 210009, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-xin Wang; Ning PanDepartment of Microbiology and Immunology, Medical School of Southeast University, 87 Dingjiaqiao Road, Nanjing, Jiangsu Province 210009, People’s Republic of ChinaEmail lxwang@seu.edu.cn; panningmicro@aliyun.comBackground 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.Keywords: ubiquitinated proteins, alumina nanoparticles, cancer vaccine, combination therapy