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Abstract
The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4+ T, CD8+ T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.

Affiliations

Ying Zhang 1, Wenzhi Tan 2, Rukhsora D Sultonova 1, Dinh-Huy Nguyen 1, Jin Hai Zheng 3, Sung-Hwan You 4, Joon Haeng Rhee 5, So-Young Kim 6, Koemchhoy Khim 5, Yeongjin Hong 7, Jung-Joon Min 8
1Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea; Department of Molecular Medicine (BrainKorea21 Plus), Chonnam National University Graduate School, Gwangju, 61469, Republic of Korea.
2School of Food Science and Bioengineering, Changsha University of Science & Technology, Changsha, Hunan, 410114, China.
3School of Biomedical Sciences, Hunan University, Changsha, Hunan, 410082, China.
4CNCure Biotech, Inc, Republic of Korea.
5Department of Microbiology, Chonnam National University Medical School, Hwasun, 58128, Republic of Korea.
6Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea.
7Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea; Department of Microbiology, Chonnam National University Medical School, Hwasun, 58128, Republic of Korea. Electronic address: yjhong@jnu.ac.kr.
8Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea; Department of Molecular Medicine (BrainKorea21 Plus), Chonnam National University Graduate School, Gwangju, 61469, Republic of Korea; Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Hwasun, 58128, Republic of Korea. Electronic address: jjmin@jnu.ac.kr.