Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy
Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR...
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MDPI AG
2021-06-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/13/11/2801 |
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| author | Ying Zhang Ramar Thangam Sung-Hwan You Rukhsora D. Sultonova Akhil Venu Jung-Joon Min Yeongjin Hong |
| author_facet | Ying Zhang Ramar Thangam Sung-Hwan You Rukhsora D. Sultonova Akhil Venu Jung-Joon Min Yeongjin Hong |
| author_sort | Ying Zhang |
| collection | DOAJ |
| description | Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from <i>E. coli</i> by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment. |
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| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-10T10:43:11Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-ad36d1e331c942e49d7f5b8cf6b0e97f2023-11-21T22:47:51ZengMDPI AGCancers2072-66942021-06-011311280110.3390/cancers13112801Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer ChemotherapyYing Zhang0Ramar Thangam1Sung-Hwan You2Rukhsora D. Sultonova3Akhil Venu4Jung-Joon Min5Yeongjin Hong6Department of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaDepartment of Nuclear Medicine, Institute for Molecular Imaging and Theranostics, Hwasun Hospital, Chonnam National University Medical School, Hwasun 58128, KoreaSurface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from <i>E. coli</i> by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.https://www.mdpi.com/2072-6694/13/11/2801monobodycalreticulinimmunogenic cell death (ICD)optical bioluminescence imagingearly prediction of chemotherapeutic response |
| spellingShingle | Ying Zhang Ramar Thangam Sung-Hwan You Rukhsora D. Sultonova Akhil Venu Jung-Joon Min Yeongjin Hong Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy Cancers monobody calreticulin immunogenic cell death (ICD) optical bioluminescence imaging early prediction of chemotherapeutic response |
| title | Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy |
| title_full | Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy |
| title_fullStr | Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy |
| title_full_unstemmed | Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy |
| title_short | Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy |
| title_sort | engineering calreticulin targeting monobodies to detect immunogenic cell death in cancer chemotherapy |
| topic | monobody calreticulin immunogenic cell death (ICD) optical bioluminescence imaging early prediction of chemotherapeutic response |
| url | https://www.mdpi.com/2072-6694/13/11/2801 |
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