홍영진 (전남대학교 의과대학)

한빛사논문

조회205

전남대학교 의과대학

Theranostics, Published 2024-1-20, 14 (3) | https://doi.org/10.7150/thno.90376 The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

Ying Zhang^1,9, Venu Akhil¹, Ho Seong Seo², Hae Ran Park², Soo Hyun Kim^3,4, Sung-Hwan You^1,5, Zhipeng Liu⁶, So-young Kim^1,5, Rukhsora D. Sultonova^1,7,8, Jung-Joon Min^1,5*, Yeongjin Hong^1,3,5*

¹Institute for Molecular Imaging and Theranostics, Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Jeollanam-do, Republic of Korea.

²Research Division for Radiation Science, Korea Atomic Energy Research Institute, Jeollabuk-do, Republic of Korea.

³Department of Microbiology, Chonnam National University Medical School, Jeollanam-do, Republic of Korea.

⁴Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Republic of Korea.

⁵CNCure Biotech, Inc., Jeollanam-do, Republic of Korea.

⁶Brain Tumor Research Laboratory, Biomedical Research Institute, Chonnam National University Hwasun Hospital, Jeollanam-do, Republic of Korea.

⁷New Uzbekistan University, Tashkent, Uzbekistan.

⁸Republican Oncology Research Center Tashkent Region Branch, Tashkent, Uzbekistan.

⁹Current affiliation: State Key Laboratory of Drug Research, Molecular Imaging Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.

Corresponding authors: Yeongjin Hong, Ph.D.

Abstract

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4 ⁺ T, and CD8 ⁺ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

논문정보

형식Research article
게재일2024년 01월 (BRIC 등록일 2024-02-14)
연구진국내(교신)+국외 연구진
분야 의약학 > 응용의학

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