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Abstract
L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

Affiliations

Ying Zhang 1, Rukhsora D Sultonova 1, Sung-Hwan You 1, Yoonjoo Choi 2, So-Young Kim 1, Wan-Sik Lee 3, Jihyoun Seong 4, Jung-Joon Min 5, Yeongjin Hong 6
1Institute for Molecular Imaging and Theranostics, Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Hwasun, Republic of Korea.
2Combinatorial Tumor Immunotherapy MRC, Chonnam National University Medical School, Hwasun, Republic of Korea.
3Department of Internal Medicine, Chonnam National University Medical School, Hwasun, Republic of Korea.
4Institute for Molecular Imaging and Theranostics, Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Hwasun, Republic of Korea; Department of Microbiology, Chonnam National University Medical School, Hwasun, Republic of Korea.
5Institute for Molecular Imaging and Theranostics, Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Hwasun, Republic of Korea. Electronic address: jjmin@jnu.ac.kr.
6Institute for Molecular Imaging and Theranostics, Department of Nuclear Medicine, Chonnam National University Medical School and Hwasun Hospital, Hwasun, Republic of Korea; Department of Microbiology, Chonnam National University Medical School, Hwasun, Republic of Korea. Electronic address: yjhong@jnu.ac.kr.