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1
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Zhou DD, Zhai XT, Zhang LW, Xie ZH, Wang Y, Zhen YS, Gao RJ, Miao QF. A new TROP2-targeting antibody-drug conjugate shows potent antitumor efficacy in breast and lung cancers. NPJ Precis Oncol 2024; 8:94. [PMID: 38654141 DOI: 10.1038/s41698-024-00584-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Accepted: 03/28/2024] [Indexed: 04/25/2024] Open
Abstract
Trophoblast cell surface antigen 2 (Trop2) is considered to be an attractive therapeutic target in cancer treatments. We previously generated a new humanized anti-Trop2 antibody named hIMB1636, and designated it as an ideal targeting carrier for cancer therapy. Lidamycin (LDM) is a new antitumor antibiotic, containing an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). AE and LDP can be separated and reassembled, and the reassembled LDM possesses cytotoxicity similar to that of native LDM; this has made LDM attractive in the preparation of gene-engineering drugs. We herein firstly prepared a new fusion protein hIMB1636-LDP composed of hIMB1636 and LDP by genetic engineering. This construct showed potent binding activities to recombinant antigen with a KD value of 4.57 nM, exhibited binding to Trop2-positive cancer cells and internalization and transport to lysosomes, and demonstrated powerful tumor-targeting ability in vivo. We then obtained the antibody-drug conjugate (ADC) hIMB1636-LDP-AE by molecular reconstitution. In vitro, hIMB1636-LDP-AE inhibited the proliferation, migration, and tumorsphere formation of tumor cells with half-maximal inhibitory concentration (IC50) values at the sub-nanomolar level. Mechanistically, hIMB1636-LDP-AE induced apoptosis and cell-cycle arrest. In vivo, hIMB1636-LDP-AE also inhibited the growth of breast and lung cancers in xenograft models. Moreover, compared to sacituzumab govitecan, hIMB1636-LDP-AE showed more potent antitumor activity and significantly lower myelotoxicity in tumors with moderate Trop2 expression. This study fully revealed the potent antitumor efficacy of hIMB1636-LDP-AE, and also provided a new preparation method for LDM-based ADC, as well as a promising candidate for breast cancer and lung cancer therapeutics.
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Affiliation(s)
- Dan-Dan Zhou
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Xiao-Tian Zhai
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Lan-Wen Zhang
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Zi-Hui Xie
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Ying Wang
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Yong-Su Zhen
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Rui-Juan Gao
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
| | - Qing-Fang Miao
- NHC Key Laboratory of Biotechnology for Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
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He S, Zhao C, Tao H, Sheng W, Gao R, Liu X, Zhen Y. A recombinant scFv antibody-based fusion protein that targets EGFR associated with IMPDH2 downregulation and its drug conjugate show therapeutic efficacy against esophageal cancer. Drug Deliv 2022; 29:1243-1256. [PMID: 35416106 PMCID: PMC9048960 DOI: 10.1080/10717544.2022.2063454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
The present study aimed to evaluate the anti-tumor efficacy of the epidermal growth factor receptor (EGFR)-targeting recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugate Fv-LDP-D3-AE against esophageal cancer. Fv-LDP-D3, consisting of the fragment variable (Fv) of an anti-EGFR antibody, the apoprotein of lidamycin (LDP), and the third domain of human serum albumin (D3), exhibited a high binding affinity for EGFR-overexpressing esophageal cancer cells, inhibited EGFR phosphorylation and down-regulated inosine monophosphate dehydrogenase type II (IMPDH2) expression. Fv-LDP-D3 was taken up by cancer cells through intensive macropinocytosis; it inhibited the proliferation and induced the apoptosis of esophageal cancer cells. In vivo imaging revealed that Fv-LDP-D3 displayed specific tumor-site accumulation and a long-lasting retention over a 26-day period. Furthermore, Fv-LDP-D3-AE, a pertinent antibody-drug conjugate prepared by integrating the enediyne chromophore of lidamycin into the Fv-LDP-D3 molecule, displayed highly potent cytotoxicity, inhibited migration and invasion, induced apoptosis and DNA damage, arrested cells at G2/M phase, and caused mitochondrial damage in esophageal cancer cells. More importantly, both of Fv-LDP-D3 and Fv-LDP-D3-AE markedly inhibited the growth of esophageal cancer xenografts in athymic mice at well tolerated doses. The present results indicate that Fv-LDP-D3, and Fv-LDP-D3-AE exert prominent antitumor efficacy associated with targeting EGFR, suggesting their potential as promising candidates for targeted therapy against esophageal cancer.
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Affiliation(s)
- Shiming He
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Chunyan Zhao
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Hongyu Tao
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Weijin Sheng
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Ruijuan Gao
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Xiujun Liu
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Yongsu Zhen
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
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3
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Gong J, Guo F, Cheng W, Fan H, Miao Q, Yang J. Preliminary biological evaluation of 123I-labelled anti-CD30-LDM in CD30-positive lymphomas murine models. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2020; 48:408-414. [PMID: 31913714 DOI: 10.1080/21691401.2019.1709857] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Accepted: 09/19/2019] [Indexed: 01/21/2023]
Abstract
Overexpression of CD30 has been reported on the surface of some T-cell lymphomas, especially on Hodgkin's lymphoma (HL) and anaplastic large cell lymphoma (ALCL). CD30 targeted immunotherapy has good clinical therapy response. We have produced a novel antibody drug conjugates (ADCs)-anti-CD30-LDM, which shows attractive tumour-targeting capability and extremely potent antitumor efficacy. To further investigate biological characteristics and promote clinical translation of anti-CD30-LDM, we constructed a radiolabeled 123I-anti-CD30-LDM to evaluate the biodistribution characteristics. The anti-CD30-LDM was radioiodinated by the Iodogen method. The radiochemical purity of 123I-anti-CD30-LDM was more over 98%, and the specific activity of 240.5 MBq/mg. The stability and the specificity of 123I-anti-CD30-LDM were evaluated in vitro. Cellular binding assays were used to evaluate the binding capabilities in CD30-positive Karpas299 cells and CD30-negative Raji cells. B-NDG mice bearing Karpas 299 and Raji xenografts were used for in vivo biodistribution studies. Our results demonstrated that anti-CD30-LDM as an ideal ADC targeted to CD30, which was labelled easily with 123I and obtained the sufficient yields. The 123I-anti-CD30-LDM preserved specific binding to CD30 in vitro and uptake in tumour xenografts in B-NDG mice. These results are encouraging for anti-CD30-LDM as a promising clinical translational candidate for various CD30 positive lymphomas and other diseases.
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MESH Headings
- Animals
- Antineoplastic Agents, Immunological/chemistry
- Antineoplastic Agents, Immunological/pharmacokinetics
- Antineoplastic Agents, Immunological/pharmacology
- Humans
- Iodine Radioisotopes/chemistry
- Iodine Radioisotopes/pharmacokinetics
- Iodine Radioisotopes/pharmacology
- Ki-1 Antigen/antagonists & inhibitors
- Lymphoma, Large-Cell, Anaplastic/drug therapy
- Lymphoma, Large-Cell, Anaplastic/metabolism
- Lymphoma, Large-Cell, Anaplastic/pathology
- Mice
- Neoplasm Proteins/antagonists & inhibitors
- Neoplasms, Experimental/drug therapy
- Neoplasms, Experimental/metabolism
- Neoplasms, Experimental/pathology
- Radiopharmaceuticals/chemistry
- Radiopharmaceuticals/pharmacokinetics
- Radiopharmaceuticals/pharmacology
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Jianhua Gong
- NHC Key Laboratory of Biotechnology of Antibiotics, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Feihu Guo
- High Tech of Atom Co. Ltd, Beijing, China
| | | | | | - Qingfang Miao
- NHC Key Laboratory of Biotechnology of Antibiotics, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Jigang Yang
- Nuclear Medicine Department, Beijing Friendship Hospital, Affiliated to Capital Medical University, Beijing, China
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4
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Wang R, Li L, Zhang S, Li Y, Wang X, Miao Q, Zhen Y. A novel enediyne-integrated antibody-drug conjugate shows promising antitumor efficacy against CD30 + lymphomas. Mol Oncol 2018; 12:339-355. [PMID: 29316337 PMCID: PMC5830626 DOI: 10.1002/1878-0261.12166] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 12/25/2017] [Accepted: 12/26/2017] [Indexed: 01/26/2023] Open
Abstract
CD30 is a 120-kDa type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily. Overexpression of CD30 has been reported in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). CD30-targeted treatment with antibody-drug conjugates (ADCs) can lead to promising clinical benefit. Lidamycin (LDM), consisting of an apoprotein LDP and an active enediyne chromophore AE, is a member of the enediyne antibiotic family and one of the most potent antitumor agents. AE and LDP can be dissociated and reconstituted under certain conditions in vitro. LDM is an ideal payload for the preparation of ADCs. In this study, we show the generation, production, and antitumor activity of anti-CD30-LDM, a novel ADC which consists of the intact anti-CD30 antibody and LDM. First, the anti-CD30-LDP fusion protein was constructed and expressed in CHO/dhFr- cells. Anti-CD30-LDP showed specific and high-affinity binding to CD30 and could be internalized into target cells. It also exhibited excellent tumor-targeting capability in vivo. Next, anti-CD30-LDM was prepared by assembling the enediyne molecule AE to the fusion protein anti-CD30-LDP. Anti-CD30-LDM was highly cytotoxic to HL and ALCL cell lines, with IC50 values of 5-50 pm. It can also induce cell apoptosis and G2/M cell cycle arrest. In the Karpas299 xenograft model, the tumor growth was inhibited by 87.76% in mice treated with anti-CD30-LDM and with no discernible adverse effects. Taken together, anti-CD30-LDM shows attractive tumor-targeting capability and antitumor efficacy both in vitro and in vivo and could be a promising candidate for the treatment of CD30+ lymphomas.
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Affiliation(s)
- Rong Wang
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Liang Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Shenghua Zhang
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Yi Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Xiaofei Wang
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Qingfang Miao
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Yongsu Zhen
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
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Yang JL, Qin Y, Li L, Cao CY, Wang Q, Li Q, Lv YF, Wang Y. Apoptotic Melanoma B16-F1 Cells Induced by Lidamycin Could Initiate the Antitumor Immune Response in BABL/c Mice. Oncol Res 2016; 23:79-86. [PMID: 26802654 PMCID: PMC7842507 DOI: 10.3727/096504015x14478843952942] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
In the process of tumor cell apoptosis induced by specific regents, calreticulin (CRT) was transferred from endoplasmic reticulum (ER) onto the cell membrane. These tumor cells, when used as the cellular vaccine to immunize experimental animals, could initiate effective antitumor immunoresponse against homologous tumor cells. This is referred to as immunogenic cell death. Lidamycin (LDM) is an enediyne antibiotic, which has extremely potent cytotoxicity to cancer cells. In this study, the mouse melanoma B16-F1 cancer cells were used to investigate the ability of LDM in promoting immunogenic cell death. Our data showed that LDM could induce apoptosis of B16-F1 cancer cells, accompanied by CRT translocation onto the cell membrane. These LDM-treated B16-F1 cells could be recognized and phagocytosed more efficiently by macrophage and dendritic cells. When the LDM-treated apoptotic B16-F1 cells were used as a whole-cell tumor vaccine to immune mice, the mice obtained resistance against rechallenged B16-F1 living cells. At the same time, the specific antitumor immune response was observed in these vaccinated mice. The splenocytes from the mice vaccinated with LDM-treated B16-F1 cells showed significantly enhanced NK lymphocyte activities and also faster growth rate and increased secretion of IFN-γ when encountering the cellular antigens from B16-F1 cells. All these results suggested that LDM could promote immunogenic cell death in B16-F1 cells, and these LDM-treated B16-F1 cells could be used as a sort of cell vaccine to initiate effective antitumor immunoresponse in mice.
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Affiliation(s)
- Jian-lin Yang
- China Three Gorges University Medical College, Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Yichang, Hubei, China
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6
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Li W, Li X, Huang T, Teng Q, Crnovcic I, Rader C, Shen B. Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation. Bioorg Med Chem 2016; 24:3887-3892. [PMID: 27094150 DOI: 10.1016/j.bmc.2016.04.017] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2016] [Revised: 04/06/2016] [Accepted: 04/07/2016] [Indexed: 11/25/2022]
Abstract
Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.
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Affiliation(s)
- Wenli Li
- Division of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA
| | - Xiuling Li
- Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Tingting Huang
- Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Qihui Teng
- Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Ivana Crnovcic
- Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Christoph Rader
- Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, USA
| | - Ben Shen
- Division of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA; Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, USA; Natural Products Library Initiative at The Scripps Research Institute, The Scripps Research Institute, Jupiter, FL 33458, USA.
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7
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Construction of a genetically engineered chimeric apoprotein consisting of sequences derived from lidamycin and neocarzinostatin. Anticancer Drugs 2016; 27:24-8. [DOI: 10.1097/cad.0000000000000300] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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8
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HIRAMA M. Total synthesis and related studies of large, strained, and bioactive natural products. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2016; 92:290-329. [PMID: 27725470 PMCID: PMC5243947 DOI: 10.2183/pjab.92.290] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/15/2016] [Accepted: 08/10/2016] [Indexed: 06/06/2023]
Abstract
Our chemical syntheses and related scientific investigations of natural products with complex architectures and powerful biological activities are described, focusing on the very large 3 nm-long polycyclic ethers called the ciguatoxins, highly strained and labile chromoprotein antitumor antibiotics featuring nine-membered enediyne cores, and extremely potent anthelmintic macrolides called the avermectins.
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Affiliation(s)
- Masahiro HIRAMA
- Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Miyagi, Japan
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9
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Li L, Shang B, Hu L, Shao R, Zhen Y. Site-specific PEGylation of lidamycin and its antitumor activity. Acta Pharm Sin B 2015; 5:264-9. [PMID: 26579455 PMCID: PMC4629235 DOI: 10.1016/j.apsb.2015.03.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Revised: 12/18/2014] [Accepted: 12/24/2014] [Indexed: 11/22/2022] Open
Abstract
In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (Mw 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs.
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Key Words
- ADCs, antibody drug conjugates
- AE, active enediyne
- Anti-TNF Fab′, anti-tumor necrosis factor Fab′
- DMSO, dimethyl sulfoxide
- Enediyne antibiotic
- G-CSF, granulocyte colony stimulating factor
- IC50 values, half-inhibitory concentrations
- IFN, interferon
- IPTG, isopropyl-β-d-thiogalactoside
- LB, Luria-Bertani
- LDM, lidamycin
- Lidamycin
- PEG, polyethyleneglycol
- Polyethylene glycol
- SEC-HPLC, size-exclusion high-performance liquid chromatography
- Site-specific PEGylation
- mPEG-ALD, methoxy-PEG-propionaldehyde
- rLDP, recombinant lidamycin apoprotein
- rhG-CSF, recombinant human granulocyte colony stimulating factor
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Affiliation(s)
| | | | | | - Rongguang Shao
- Corresponding authors. Tel.: +86 10 63026956; fax: +86 10 63017302.
| | - Yongsu Zhen
- Corresponding authors. Tel.: +86 10 63026956; fax: +86 10 63017302.
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10
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Gao R, Li L, Shang B, Zhao C, Sheng W, Li D. A Gelatinases-targeting scFv-based Fusion Protein Shows Enhanced Antitumour Activity with Endostar against Hepatoma. Basic Clin Pharmacol Toxicol 2015; 117:105-16. [PMID: 25615234 DOI: 10.1111/bcpt.12379] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Accepted: 01/09/2015] [Indexed: 12/26/2022]
Abstract
Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours.
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Affiliation(s)
- Ruijuan Gao
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Liang Li
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Boyang Shang
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Chunyan Zhao
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Weijin Sheng
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Diandong Li
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
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11
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Liu WJ, Liu XJ, Li L, Li Y, Zhang SH, Zhen YS. Tuftsin-based, EGFR-targeting fusion protein and its enediyne-energized analog show high antitumor efficacy associated with CD47 down-regulation. Cancer Immunol Immunother 2014; 63:1261-72. [PMID: 25164878 PMCID: PMC11029470 DOI: 10.1007/s00262-014-1604-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2013] [Accepted: 08/15/2014] [Indexed: 01/13/2023]
Abstract
Tuftsin (TF) is an immunomodulator tetrapeptide (Thr-Lys-Pro-Arg) that binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Many reports have described anti-tumor activity of tuftsin to relate with nonspecific activation of the host immune system. Lidamycin (LDM) that displays extremely potent cytotoxicity to cancer cells is composed of an apoprotein (LDP) and an enediyne chromophore (AE). In addition, Ec is an EGFR-targeting oligopeptide. In the present study, LDP was used as protein scaffold and the specific carrier for the highly potent AE. Genetically engineered fusion proteins LDP-TF and Ec-LDP-TF were prepared; then, the enediyne-energized fusion protein Ec-LDM-TF was generated by integration of AE into Ec-LDP-TF. The tuftsin-based fusion proteins LDP-TF and Ec-LDP-TF significantly enhanced the phagocytotic activity of macrophages as compared with LDP (P < 0.05). Ec-LDP-TF effectively bound to tumor cells and macrophages; furthermore, it markedly suppressed the growth of human epidermoid carcinoma A431 xenograft in athymic mice by 84.2 % (P < 0.05) with up-regulated expression of TNF-α and IFN-γ. Ec-LDM-TF further augmented the therapeutic efficacy, inhibiting the growth of A431 xenograft by 90.9 % (P < 0.05); notably, the Ec-LDM-TF caused marked down-regulation of CD47 in A431 cells. Moreover, the best therapeutic effect was recorded in the group of animals treated with the combination of Ec-LDP-TF with Ec-LDM-TF. The results suggest that tuftsin-based, enediyne-energized, and EGFR-targeting fusion proteins exert highly antitumor efficacy with CD47 modulation. Tuftsin-based fusion proteins are potentially useful for treatment of EGFR- and CD47-overexpressing cancers.
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Affiliation(s)
- Wen-Juan Liu
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
- Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan, 250117 Shandong China
| | - Xiu-Jun Liu
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
| | - Liang Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
| | - Yi Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
| | - Sheng-Hua Zhang
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
| | - Yong-Su Zhen
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1, Tiantan Xili, Beijing, 100050 China
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12
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Li B, Zheng YB, Li DD, Zhen YS. Preparation and evaluation of a CD13/APN-targeting and hydrolase-resistant conjugate that comprises pingyangmycin and NGR motif-integrated apoprotein. J Pharm Sci 2014; 103:1204-13. [PMID: 24504597 DOI: 10.1002/jps.23893] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2013] [Revised: 12/31/2013] [Accepted: 01/17/2014] [Indexed: 11/10/2022]
Abstract
We have chemically synthesized NGR-LDP-PYM, a novel CD13/aminopeptidase (APN)-targeting and hydrolase-resistant conjugate by cross-linking of the antitumor antibiotic pingyangmycin (bleomycin A5 , PYM) to an engineered NGR motif-integrated apoprotein (NGR-LDP) with a noncleavable linker. This protein-drug conjugate not only basically retains the original properties of PYM but also can specifically deliver PYM to the CD13/APN-expressing tumor cells. Furthermore, the resulting conjugate exhibits more resistance to hydrolysis of recombinant human bleomycin hydrolase than parental PYM. These results may be useful for improving the therapeutic efficacy of PYM and have implications in the treatment of PYM-refractory and CD13/APN-overexpressing tumors.
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Affiliation(s)
- Bin Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
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13
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Chemiluminescent detection of cell apoptosis enzyme by gold nanoparticle-based resonance energy transfer assay. Anal Bioanal Chem 2014; 406:5677-84. [PMID: 24481623 DOI: 10.1007/s00216-013-7611-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2013] [Revised: 12/27/2013] [Accepted: 12/30/2013] [Indexed: 10/25/2022]
Abstract
We report a new chemiluminescence resonance energy transfer (CRET) technique, using gold nanoparticles (AuNPs) as efficient energy acceptor, for homogeneous measurement of cell apoptosis enzyme with high sensitivity. In the design of the CRET system, we chose the highly sensitive chemiluminescence (CL) reaction between luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λ max 425 nm) partially overlaps the visible absorption bands of AuNPs. In this system, the peptide substrate (DEVD) of caspase 3 was linked to the AuNP surface by Au-S linkage. HRP was attached to the AuNP surface by means of a bridge formed by the streptavidin-biotin reaction. CRET occurred as a result of formation of AuNP-peptide-biotin-streptavidin-HRP complexes. The CL of luminol was significantly reduced, because of the quenching effect of AuNPs. The quenched CL was recovered after cleavage of DEVD by caspase 3, an enzyme involved in the apoptotic process. Experimental conditions were systematically investigated. Under the optimum conditions the increase of the CL signal was linearly dependent on caspase 3 concentration within the concentration range 25 pmol L(-1) to 800 pmol L(-1) and the detection limit of caspase 3 was as low as 20 pmol L(-1), one order of magnitude lower than for FRET sensors based on graphene oxides. Our method was successfully used to detect drug-induced apoptosis of cells. This approach is expected to be extended to other assays, i.e., using other enzymes, analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene).
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14
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Li B, Liu XJ, Li L, Zhang SH, Li Y, Li DD, Zhen YS. A tumor-targeting dextran–apoprotein conjugate integrated with enediyne chromophore shows highly potent antitumor efficacy. Polym Chem 2014. [DOI: 10.1039/c4py00532e] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
A novel dextran–apoprotein conjugate that could selectively stay in tumor tissues for a prolonged time was prepared. After integrating with enediyne chromophore, this conjugate showed highly potent antitumor efficacy.
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Affiliation(s)
- Bin Li
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Xiu-jun Liu
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Liang Li
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Sheng-hua Zhang
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Yi Li
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Dian-dong Li
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
| | - Yong-su Zhen
- Institute of Medicinal Biotechnology
- Chinese Academy of Medical Sciences and Peking Union Medical College
- Beijing 100050, China
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15
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Increasing appendage diversity on 3,4-dihydro-3-oxo-2H-1,4-benzoxazines via Aphos–Pd(OAc)2-catalyzed Suzuki–Miyaura cross-coupling of aryl chlorides. Tetrahedron 2013. [DOI: 10.1016/j.tet.2013.09.057] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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16
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Ruan L, Xu Z, Lan T, Wang J, Liu H, Li C, Dong C, Ren J. Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy. Anal Chem 2012; 84:7350-8. [PMID: 22876965 DOI: 10.1021/ac301654g] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.
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Affiliation(s)
- Lingao Ruan
- College of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiaotong University, Shanghai 200240, People's Republic of China
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17
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Zhang Q, Liu X, Xu S, Li C, Zhang Y, Yang J, Zheng J. Factor VII light chain-targeted lidamycin shows intensified therapeutic efficacy for liver cancer. Cancer Biother Radiopharm 2012; 27:384-91. [PMID: 22651685 DOI: 10.1089/cbr.2012.1209] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC(50)) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients.
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Affiliation(s)
- Qing Zhang
- Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, PR China.
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18
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Releasing of the chromophore from the drug delivery protein C-1027: A molecular dynamics simulations study. J Struct Biol 2010; 172:284-93. [DOI: 10.1016/j.jsb.2010.08.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2010] [Revised: 08/12/2010] [Accepted: 08/18/2010] [Indexed: 11/19/2022]
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19
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Inhibition of mouse embryonic carcinoma cell growth by lidamycin through down-regulation of embryonic stem cell-like genes Oct4, Sox2 and Myc. Invest New Drugs 2010; 29:1188-97. [PMID: 20596749 DOI: 10.1007/s10637-010-9463-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2010] [Accepted: 05/20/2010] [Indexed: 12/24/2022]
Abstract
Lidamycin (LDM, also known as C-1027) as an anti-cancer agent inhibits growth in a variety of cancer cells by inducing apoptosis and cell cycle arrest. In this study we demonstrated that inhibition of mouse embryonic carcinoma (EC) cell growth using LDM at low concentrations can be attributed to a loss of the cell's self-renewal capability but not to apoptosis or cell death, which can be correlated to the down-regulation of embryonic stem (ES) cell-like genes Oct4, Sox2 and c-Myc. MTT assays showed that LDM inhibited the growth of mouse P19 EC cells in a time- and dose-dependent manner. The EC cells exposed to a low dose (0.01 nM) of LDM lost their capability to generate colonies, as evidenced by the colony forming assay. Flow cytometer analyses demonstrated that LDM induced G1 arrest in exposed EC cells without apoptosis. Real-time qPCR, Western blotting and immunocytochemistry revealed that Oct4, Sox2 and c-Myc were down-regulated in LDM-exposed EC cells, but not adriamycin (ADM)-exposed cells. Furthermore, a combination of the low dose of LDM and ADM significantly reduced the proliferation of the cancer cells than single-agent treatment. This suggested that synergy of ADM and LDM improved chemotherapy. Taking together, our results indicate that LDM can reduce the capability for self-renewal that mouse EC cells possess through the repression of ES cell-like genes, thereby inhibiting carcinoma cell growth. This data also suggests that LDM might have potential for application in CSC-based therapy and be a useful tool for studying ES cell pluripotency and differentiation.
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20
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Xin C, Ye S, Ming Y, Shenghua Z, Qingfang M, Hongxing G, Xu S, Yuanfu X, Yuan Z, Dongmei F, Juanni L, Yingdai G, Lianfang J, Rongguang S, Zhenping Z, Jianxiang W, Tao C, Chunzheng Y, Dongsheng X, Yongsu Z. Efficient inhibition of B-cell lymphoma xenografts with a novel recombinant fusion protein: anti-CD20Fab-LDM. Gene Ther 2010; 17:1234-43. [PMID: 20463754 DOI: 10.1038/gt.2010.76] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Lidamycin (LDM) is a new member of enediyne antitumor antibiotics family that can be separated and reconstituted. It consists of a labile active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). LDM is now in phase II clinical trials. In this study, we described the antitumor features of a fusion protein of LDM, anti-CD20Fab-LDM, targeted to CD20 expressed by B-lymphoid malignancies. Especially, LDM was prepared by a novel two-step method including DNA recombination and molecular reconstitution. Anti-CD20Fab-LDM exerted potent cytotoxicity against CD20+ B-cell lymphoma cell lines in vitro (IC50: 10-30 pM) and in the Raji xenograft model. Two Raji xenografts were allowed to grow to an initial mass of 80 and 500 mm³, respectively, and then anti-CD20Fab-LDM was administered intravenously with the highest dose of 4 nmol kg⁻¹ . The inhibition rates of tumor growth were 90.1 and 85%, which were saliently superior to those of nontargeted LDM. It is noteworthy that anti-CD20Fab-LDM can inhibit the growth of patient-derived cells, including rituximab-resistant patient-derived cells. Thus, CD20-targeted delivery of LDM is a specific and potent therapeutic strategy for B-lymphoid malignancies. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs.
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Affiliation(s)
- C Xin
- State Key Laboratory of Experimental Hematology, Department of Pharmacy, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, PRC
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21
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Brucher B, Rudat J, Syldatk C, Vielhauer O. Enantioseparation of Aromatic β³-Amino acid by Precolumn Derivatization with o-Phthaldialdehyde and N-Isobutyryl-l-cysteine. Chromatographia 2010. [DOI: 10.1365/s10337-010-1578-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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22
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A tandem scFv-based fusion protein and its enediyne-energized analogue show intensified therapeutic efficacy against lung carcinoma xenograft in athymic mice. Cancer Lett 2010; 295:124-33. [PMID: 20303650 DOI: 10.1016/j.canlet.2010.02.020] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Revised: 01/28/2010] [Accepted: 02/19/2010] [Indexed: 11/22/2022]
Abstract
Gelatinases play important roles in tumor progression and are abundantly expressed in a variety of malignant tumors. Antibody targeting gelatinases is a possible avenue to fight against cancer. However, antibody alone can not achieve curative efficacy. Herein, we demonstrated the intensified targeting therapy of a tandem scFv-based fusion protein and its enediyne-energized analogue against gelatinases-overexpressed tumor. A fusion protein dFv-LDP, comprising a tandem scFv of anti-gelatinases linked to the apoprotein (LDP) of lidamycin, was generated and showed strong tumor targeting capability in three different tumor xenografts. In PG-BE1 lung carcinoma xenograft, the tumor inhibition rate was 77.5% by dFv-LDP versus 94.2% by dFv-LDP-AE, the product of dFv-LDP assembled with the active enediyne chromophore (AE) of lidamycin. Moreover, the combination of dFv-LDP with dFv-LDP-AE further augmented the therapeutic efficacy, producing initial tumor shrinkage in five of six mice. The microvessel density (P<0.05) and proliferation index (P<0.05) were also stepwise decreased in groups of dFv-LDP, dFv-LDP-AE and the combination. In conclusion, our results demonstrated that the antibody-based therapy against gelatinases was stepwise intensified in use of dFv-LDP, dFv-LDP-AE and dFv-LDP plus dFv-LDP-AE, and indicated that the combination of an antibody with its drug-armed analogue might be of interest as a new approach to augment antitumor efficacy.
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23
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Lin S, Horsman GP, Chen Y, Li W, Shen B. Characterization of the SgcF epoxide hydrolase supporting an (R)-vicinal diol intermediate for enediyne antitumor antibiotic C-1027 biosynthesis. J Am Chem Soc 2010; 131:16410-7. [PMID: 19856960 DOI: 10.1021/ja901242s] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
C-1027 is a chromoprotein antitumor antibiotic consisting of an apoprotein and the C-1027 chromophore. The C-1027 chromophore possesses four distinct structural moieties-an enediyne core, a deoxy aminosugar, a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-beta-tyrosine-the latter two of which are proposed to be appended to the enediyne core via a convergent biosynthetic strategy. Here we report the in vitro characterization of SgcF, an epoxide hydrolase from the C-1027 biosynthetic gene cluster that catalyzes regio- and stereospecific hydrolysis of styrene oxide, serving as an enediyne core epoxide intermediate mimic, to form a vicinal diol. Abolishment of C-1027 production in the DeltasgcF mutant strain Streptomyces globisporus SB1010 unambiguously establishes that sgcF plays an indispensable role in C-1027 biosynthesis. SgcF efficiently hydrolyzes (S)-styrene oxide, displaying an apparent K(m) of 0.6 +/- 0.1 mM and k(cat) of 48 +/- 1 min(-1), via attack at the alpha-position to exclusively generate the (R)-phenyl vicinal diol, consistent with the stereochemistry of the C-1027 chromophore. These findings support the role of SgcF in the proposed convergent pathway for C-1027 biosynthesis, unveiling an (R)-vicinal diol as a key intermediate. Interestingly, SgcF can also hydrolyze (R)-styrene oxide to afford preferentially the (R)-phenyl vicinal diol via attack at the beta-position, albeit with significantly reduced efficiency (apparent K(m) of 2.0 +/- 0.4 mM and k(cat) = 4.3 +/- 0.3 min(-1)). Although the latter activity unlikely contributes to C-1027 biosynthesis in vivo, such enantioconvergence arising from complementary regioselective hydrolysis of a racemic substrate could be exploited to engineer epoxide hydrolases with improved regio- and/or enantiospecificity.
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Affiliation(s)
- Shuangjun Lin
- Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222, USA
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24
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Sato I, Hirama M. Recent Advances in the Synthetic Studies of Nine-membered Enediyne Antitumor Antibiotics. J SYN ORG CHEM JPN 2010. [DOI: 10.5059/yukigoseikyokaishi.68.1123] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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25
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Liang ZX. Complexity and simplicity in the biosynthesis of enediyne natural products. Nat Prod Rep 2010; 27:499-528. [DOI: 10.1039/b908165h] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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26
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Cui Z, Wang L, Wang S, Li G, Hong B. Disruption of cagA, the apoprotein gene of chromoprotein antibiotic C-1027, eliminates holo-antibiotic production, but not the cytotoxic chromophore. FEMS Microbiol Lett 2009; 301:57-68. [PMID: 19845765 DOI: 10.1111/j.1574-6968.2009.01800.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
C-1027 is a chromoprotein of the nine-membered enediyne antitumour antibiotic family, comprising apoprotein to stabilize and transport the enediyne chromophore. The disruption of apoprotein gene cagA within the C-1027 biosynthetic gene cluster abolished C-1027 holo-antibiotic production detected by an antibacterial assay, as well as the expression of the apoprotein and C-1027 chromophore extracted following protein precipitation of the culture supernatant. Complementation of the cagA-disrupted mutant AKO with the intact cagA gene restored C-1027 production, suggesting that cagA is indispensable for holo-antibiotic production. Overexpression of cagA in the wild-type strain resulted in a significant increase in C-1027 production as expected. Surprisingly, electrospray ionization (ESI)-MS and ESI-MS/MS analyses suggested that the AKO mutant still produced the C-1027 enediyne chromophore [m/z=844 (M+H)(+)] and its aromatized product [m/z=846 (M+H)(+)]. Consistent with this, the results from gene expression analysis using real-time reverse transcriptase-PCR showed that transcripts of the positive regulator sgcR3 and the structural genes sgcA1, sgcC4, sgcD6 and sgcE were readily detected in the AKO mutant as well as in the wild-type and the complementation strain. These results provided, for the first time, evidence suggesting that the apoprotein of C-1027 is not essential in the self-resistance mechanism for the enediyne chromophore.
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Affiliation(s)
- Zhihui Cui
- Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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27
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Cai L, Chen H, Miao Q, Wu S, Shang Y, Zhen Y. Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein. J Biotechnol 2009; 144:142-50. [PMID: 19737585 DOI: 10.1016/j.jbiotec.2009.09.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2009] [Revised: 08/16/2009] [Accepted: 09/01/2009] [Indexed: 10/20/2022]
Abstract
The molecule of lidamycin that belongs to the chromoprotein family of antitumor antibiotics is composed of an apoprotein (LDP) and an enediyne chromophore. The enediyne moiety of the molecule is responsible for the potent cytotoxicity; however, the biological function of the apoprotein moiety, particularly its interaction with cancer cells, remains unclear. In present study, the binding capability of LDP to human tumors was detected for the first time by tissue microarray. LDP bound to various human tumors with significant difference from the corresponding normal tissues. Positive correlation between binding activity and the overexpression of VEGF and EGFR was confirmed by lung carcinoma tissue microarray. A fusion protein LG-LDP that consists of LDP and a ligand oligopeptide to EGFR was constructed by DNA recombination. LG-LDP showed augmented binding to EGFR-overexpressing cancer cells. Furthermore, an energized fusion protein LG-LDP-AE was prepared by integrating the active enediyne (AE) into LG-LDP molecule. By MTT assay, LG-LDP-AE displayed extremely potent cytotoxicity to cancer cells with IC50 approximate to 0.01nM. The results indicate that LDP binds to various human tumors and it might serve as a delivery carrier by integration of ligand oligopeptide to manufacture motif-based, targeted fusion proteins for cancer.
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Affiliation(s)
- Lin Cai
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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28
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Zhen YZ, Lin YJ, Li Y, Zhen YS. Lidamycin shows highly potent cytotoxic to myeloma cells and inhibits tumor growth in mice. Acta Pharmacol Sin 2009; 30:1025-32. [PMID: 19575006 DOI: 10.1038/aps.2009.75] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
AIM To investigate the effects of lidamycin (LDM) on a mouse myeloma cell line (SP2/0) and human multiple myeloma cell lines (U266 and SKO-007), and provide the basis for the use of LDM in cancer therapy. METHODS A 3-[4,5-dimethylthiazol-2-yl]5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]2H-tetrazolium inner salt (MTS) assay was used to determine the degree of growth inhibition by the drugs analyzed in this study. Cell cycle distribution and analysis were measured by flow cytometry combined with propidium iodide (PI) staining. The effects on apoptosis were measured by Hoechst 33342 staining and by flow cytometry combined with fluorescein-isothiocyanate-Annexin V/propidium iodide (FITC-Annexin V/PI) staining. Protein expression was determined by Western blot analysis. In vivo antitumor activity was measured using a murine myeloma model in BALB/c mice. RESULTS There was a significant reduction in cell proliferation after treatment with LDM. The overall growth inhibition correlated with increased apoptotic cell death. LDM-induced cell apoptosis was associated with the activation of c-Jun-N-terminal kinase (JNK), and cleavage of caspase-3/7 and poly (ADP-ribose) polymerase (PARP). LDM markedly suppressed tumor growth in a murine myeloma model. CONCLUSION LDM induces apoptosis in murine myeloma SP2/0 cells as well as in human myeloma U266 and SKO-007 cell lines. The sustained activation of JNK might play a critical role in LDM-induced apoptosis in the SP2/0 cell line. LDM demonstrates significant antitumor efficacy against myeloma SP2/0 cells in mice. Taken together, our data provide some clues for further research of the effects of LDM on human multiple myeloma.Acta Pharmacologica Sinica (2009) 30: 1025-1032; doi: 10.1038/aps.2009.75.
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29
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Enediyne lidamycin enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib, in epidermoid carcinoma A431 cells and lung carcinoma H460 cells. Anticancer Drugs 2009; 20:41-9. [PMID: 19342999 DOI: 10.1097/cad.0b013e328318292c] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Gefitinib, a low-molecular-weight epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is effective in a wide variety of tumor types. Preclinical studies have shown potentiated antitumor efficacies of this agent in combination with chemotherapy or radiotherapy. The antitumor antibiotic lidamycin (LDM) showed extremely potent cytotoxicity in vitro and marked therapeutic effect in vivo. In this report, the cytotoxic and biochemical activity of LDM and gefitinib on human epidermoid carcinoma A431 cells and human large cell lung cancer H460 cells as a single agent or in combination has been evaluated. In the MTT assay, LDM showed much more potent cytotoxicity than gefitinib to both cell lines. A431 cells with a highly EGFR-expressing level were more sensitive to gefitinib than H460 cells, which expressed EGFR at an intermediate level. LDM plus gefitinib showed potentiation of antiproliferative activity and apoptosis induction, which were associated with downregulation of EGFR signaling pathway and nuclear factor-kappa B expression, and the increase of cleaved poly (adenosine diphosphate-ribose) polymerase in the two cell lines, although to a lesser degree in H460 cells. Combined treatment induced G1 phase arrest similar to that of gefitinib alone in A431 cells and intensified G2/M phase accumulation in H460 cells. The above results indicate that LDM potentiates the effects of gefitinib in both gefitinib sensitive and less sensitive cells in association with enhanced inhibition of EGFR-dependent signaling.
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30
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Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase. Int J Hematol 2009; 90:44-51. [DOI: 10.1007/s12185-009-0340-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2008] [Revised: 03/25/2009] [Accepted: 04/23/2009] [Indexed: 10/20/2022]
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31
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Gao M, Skolnick J. DBD-Hunter: a knowledge-based method for the prediction of DNA-protein interactions. Nucleic Acids Res 2008; 36:3978-92. [PMID: 18515839 PMCID: PMC2475642 DOI: 10.1093/nar/gkn332] [Citation(s) in RCA: 121] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The structures of DNA–protein complexes have illuminated the diversity of DNA–protein binding mechanisms shown by different protein families. This lack of generality could pose a great challenge for predicting DNA–protein interactions. To address this issue, we have developed a knowledge-based method, DNA-binding Domain Hunter (DBD-Hunter), for identifying DNA-binding proteins and associated binding sites. The method combines structural comparison and the evaluation of a statistical potential, which we derive to describe interactions between DNA base pairs and protein residues. We demonstrate that DBD-Hunter is an accurate method for predicting DNA-binding function of proteins, and that DNA-binding protein residues can be reliably inferred from the corresponding templates if identified. In benchmark tests on ∼4000 proteins, our method achieved an accuracy of 98% and a precision of 84%, which significantly outperforms three previous methods. We further validate the method on DNA-binding protein structures determined in DNA-free (apo) state. We show that the accuracy of our method is only slightly affected on apo-structures compared to the performance on holo-structures cocrystallized with DNA. Finally, we apply the method to ∼1700 structural genomics targets and predict that 37 targets with previously unknown function are likely to be DNA-binding proteins. DBD-Hunter is freely available at http://cssb.biology.gatech.edu/skolnick/webservice/DBD-Hunter/.
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Affiliation(s)
- Mu Gao
- Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, 250 14th Street NW, Atlanta, GA 30318, USA
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Chen J, Wu SY, Ou-Yang ZG, Zhen YS. Synergy of gemcitabine and lidamycin associated with NF-kappaB downregulation in pancreatic carcinoma cells. Acta Pharmacol Sin 2008; 29:614-9. [PMID: 18430374 DOI: 10.1111/j.1745-7254.2008.00774.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
AIM To investigate the effects on human pancreatic cancer PANC-1 and SW1990 cells using a combination of lidamycin (LDM) and gemcitabine. METHODS A 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibition of drugs in PANC-1 and SW1990 cells. The effects on apoptosis were measured by terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry combined with fluorescein- isothiocyanate-Annexin V/propidium iodide staining. The activity of caspase-3 was measured with a special assay kit. The mitochondrial membrane potential was determined by confocal microscopy analyses. The level of mRNA encoding K-ras in the cells was determined by RT-PCR analysis. The expression of K-ras, NF-kappaB, and Bcl-2 was detected by Western blotting analysis. RESULTS There was a significant reduction in proliferation in the pancreatic cancer cell lines treated with a combination of gemcitabine and LDM. The overall growth inhibition directly correlated with apoptotic cell death. LDM potentiated the gemcitabine-induced cell killing by reducing mitochondrial membrane potential and increasing the caspase-3 activity. Notably, the K-ras mRNA level was significantly reduced with the combination of gemcitabine and LDM. The results for K-ras, NF-kappaB, and Bcl-2 proteins also showed downregulation in the combination group relative to the single-agent treatment and the untreated control. CONCLUSION LDM can potentiate the growth inhibition induced by gemcitabine in human pancreatic cancer cells, and the synergy may be associated with NF-kappaB downregulation.
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Affiliation(s)
- Jing Chen
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
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Lin S, Van Lanen SG, Shen B. Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027. J Am Chem Soc 2008; 130:6616-23. [PMID: 18426211 DOI: 10.1021/ja710601d] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate.
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Affiliation(s)
- Shuangjun Lin
- Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222, USA
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Miao Q, Shang B, Ouyang Z, Liu X, Zhen Y. Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin. ACTA ACUST UNITED AC 2007; 50:447-56. [PMID: 17653664 DOI: 10.1007/s11427-007-0058-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2006] [Accepted: 02/13/2007] [Indexed: 10/23/2022]
Abstract
Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size compared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC(50) values of 8.55 x 10(-12) and 1.70 x 10(-11) mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.
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Affiliation(s)
- QingFang Miao
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100050, China
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35
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Baker JR, Woolfson DN, Muskett FW, Stoneman RG, Urbaniak MD, Caddick S. Protein–Small Molecule Interactions in Neocarzinostatin, the Prototypical Enediyne Chromoprotein Antibiotic. Chembiochem 2007; 8:704-17. [PMID: 17451164 DOI: 10.1002/cbic.200600534] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The enediyne chromoproteins are a class of potent antitumour antibiotics comprising a 1:1 complex of a protein and a noncovalently bound chromophore. The protein is required to protect and transport the highly labile chromophore, which acts as the cytotoxic component by reacting with DNA leading to strand cleavage. A derivative of the best-studied member of this class, neocarzinostatin (NCS), is currently in use as a chemotherapeutic in Japan. The application of the chromoproteins as therapeutics along with their unique mode of action has prompted widespread interest in this area. Notable developments include the discovery of non-natural ligands for the apoproteins and the observation that multiple binding modes are available for these ligands in the binding site. Mutation studies on the apoproteins have revealed much about their stability and variability, and the application of an in vitro evolution method has conferred new binding specificity for unrelated ligands. These investigations hold great promise for the application of the apoproteins for drug-delivery, transport and stabilisation systems.
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Affiliation(s)
- James R Baker
- University College London, Department of Chemistry, Christopher Ingold Laboratories, 20 Gordon Street, London, WC1H 0AJ, UK
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36
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Miao QF, Liu XY, Shang BY, Ouyang ZG, Zhen YS. An enediyne-energized single-domain antibody-containing fusion protein shows potent antitumor activity. Anticancer Drugs 2007; 18:127-37. [PMID: 17159599 DOI: 10.1097/cad.0b013e3280112779] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Single-domain antibodies are attractive as tumor-targeting vehicles because of their much smaller size than intact antibody molecules. Lidamycin is a macromolecular antitumor antibiotic, which consists of a labile enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). An enediyne-energized fusion protein VH-LDP-AE composed of single-domain antibody directed against type IV collagenase and lidamycin was prepared by a novel two-step method including DNA recombination and molecular reconstitution. VH-LDP-AE demonstrated extremely potent cytotoxicity to cancer cells and marked antiangiogenic activity in vitro. In the mouse hepatoma 22 model, drugs were administered intravenously as a single dose on day 1 with maximal tolerated doses. VH-LDP-AE (0.25 mg/kg) suppressed the tumor growth by 95.9%, whereas lidamycin (0.05 mg/kg) and mitomycin (1 mg/kg) by 79.6 and 51.1%, respectively. In the HT-1080 xenograft model in nude mice, drugs were given intravenously as a single dose on day 4 after tumor implantation. VH-LDP-AE at 0.25 mg/kg suppressed tumor growth by 76% (P<0.05) compared with that of lidamycin at 0.05 mg/kg (53%) on day 18. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein VH-LDP-AE was more effective than lidamycin and mitomycin. These properties, together with its much smaller size than conventional antibody-based agents, suggested that VH-LDP-AE would be a promising candidate for cancer-targeting therapy. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.
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Affiliation(s)
- Qing-fang Miao
- Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, #1 Tiantan Xili, Beijing 100050, PRC
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Inoue M, Usuki T, Lee N, Hirama M, Tanaka T, Hosoi F, Ohie S, Otani T. Antitumor Enediyne Chromoprotein C-1027: Mechanistic Investigation of the Chromophore-Mediated Self-Decomposition Pathway. J Am Chem Soc 2006; 128:7896-903. [PMID: 16771503 DOI: 10.1021/ja060724w] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
C-1027 is an extremely potent antitumor agent that causes double-stranded DNA cleavages. It is a unique small molecule-protein complex composed of a highly reactive enediyne chromophore, which upon binding reacts with its target molecule DNA through radical-mediated hydrogen abstraction and an apoprotein that encapsulates the chromophore serving as its carrier to reach DNA. Although C-1027 has favorable properties as an effective drug delivery system, it slowly self-decomposes due to the reactivity of the chromophore toward the apoprotein. Understanding how the C-1027 destroys itself may enable design of its analogues that overcome this limitation. In this paper, mechanistic insights into the self-reactivity of C-1027 that facilitates its own decomposition are described. We provide evidence that the formation of the Gly96 radical, which promotes the oxidative protein scission and the subsequent chromophore release, is the major pathway for the self-decomposition of C-1027. On the basis of the newly isolated products of the self-decomposition, we propose that the apoprotein effectively protects two different structural elements of the chromophore that are essential for its biological activity: the nine-membered enediyne moiety (necessary for DNA cleavage) and the benzoxazine moiety (necessary for DNA intercalation). Using an engineered apoprotein analogue kinetically more stable toward the chromophore radical, we show that enhanced overall properties can be achieved for the natural C-1027 with respect to stability and antitumor activities. The results present the first example of a rationally designed C-1027 analogue reported to display superior in vitro antitumor activity to the natural C-1027. Our findings may have implications for design of proteins that can stably encapsulate highly reactive small molecules.
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Affiliation(s)
- Masayuki Inoue
- Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan.
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Inoue M. Exploring the Chemistry and Biology of Antitumor Enediyne Chromoprotein C-1027. BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 2006. [DOI: 10.1246/bcsj.79.501] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Usuki T, Inoue M, Akiyama K, Hirama M. ESR studies on DNA cleavage induced by enediyne C-1027 chromophore. Bioorg Med Chem 2005; 13:5218-24. [PMID: 15979878 DOI: 10.1016/j.bmc.2005.05.049] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2005] [Revised: 05/26/2005] [Accepted: 05/26/2005] [Indexed: 11/23/2022]
Abstract
C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.
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Affiliation(s)
- Toyonobu Usuki
- Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan
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Takashima H, Yoshida T, Ishino T, Hasuda K, Ohkubo T, Kobayashi Y. Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein. J Biol Chem 2005; 280:11340-6. [PMID: 15640161 DOI: 10.1074/jbc.m411579200] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.
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Affiliation(s)
- Hiroyuki Takashima
- Informatics and Knowledge Management at Novartis Institutes for BioMedical Research, Novartis, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
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Liu YP, Li QS, Huang YR, Zhou MJ, Liu CX. Pharmacokinetics of C-1027 in mice as determined by TCA-RA method. World J Gastroenterol 2005; 11:717-20. [PMID: 15655829 PMCID: PMC4250746 DOI: 10.3748/wjg.v11.i5.717] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/21/2004] [Revised: 02/24/2004] [Accepted: 03/18/2004] [Indexed: 02/06/2023] Open
Abstract
AIM To validate a radioactivity assay, the TCA-RA method, for the measurement of C-1027 in serum and to evaluate its application in determination of pharmacokinetics of C-1027 in mice. METHODS (125)I-C-1027 was prepared by the Iodogen method and separated by HPLC. The radioactivity assay was established and used to determine (125)I-C-1027 in mice at doses of 10, 50 and 100 microg/kg after precipitation with 20% trichloroacetic acid (TCA-RA method). Several pharmacokinetic parameters were determined after intravenous injection of (125)I-C-1027 to mice. RESULTS After intravenous injection of (125)I-C-1027 to mice, at doses of 10, 50 and 100 microg/kg; the apparent distribution volumes (V(d)) were 0.26, 0.31 and 0.33 L/kg; the biological half-lives (T(1/2)) were 3.10, 3.40 and 3.90 h; the areas under curve (AUC) were 18.41, 103.69 and 202.74 ng/h/mL; the elimination rate constants (K) were 1.04, 1.26 and 0.58/h; and the total body clearance (Cl) were 0.54, 0.48 and 0.49 L/kg/h, respectively. CONCLUSION TCA-RA is a sensitive, reliable and suitable method for the determination of (125)I-C-1027 in mouse serum.
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Affiliation(s)
- You-Ping Liu
- Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road, Tianjin 300193, China
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Inoue M, Hatano S, Kodama M, Sasaki T, Kikuchi T, Hirama M. Synthesis of the Bicyclo[7.3.0]dodecatrienediyne Core of the C-1027 Chromophore. Org Lett 2004; 6:3833-6. [PMID: 15469361 DOI: 10.1021/ol048494i] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
[reaction: see text] C-1027, an extremely potent antitumor agent, is composed of chromophore 1 and an apoprotein. Here we report a general and efficient route to the exceedingly reactive bicyclo[7.3.0]dodecatrienediyne core of 1, utilizing selenoxide elimination and epoxide deoxygenation to build the cyclopentadiene and enediyne structures, respectively.
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Affiliation(s)
- Masayuki Inoue
- Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan.
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Usuki T, Inoue M, Hirama M, Tanaka T. Rational design of a supra C-1027: kinetically stabilized analogue of the antitumor enediyne chromoprotein. J Am Chem Soc 2004; 126:3022-3. [PMID: 15012111 DOI: 10.1021/ja039635z] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
C-1027, an extremely potent antitumor agent, is composed of a highly reactive chromophore and an apoprotein. While the chromophore causes DNA cleavage, the apoprotein functions as its carrier. Despite these ideal properties as an anticancer agent, C-1027 slowly self-decomposes through chromophore-mediated abstraction of hydrogens from the apoprotein. In this paper, we report the design and preparation of an engineered C-1027 apoprotein that decelerates this self-decomposition pathway. Our design is based on the kinetic isotope effect, and deuterium is incorporated instead of protium into the hydrogen-abstraction site. The deuterated supra C-1027 was found to have a 4-fold longer lifetime than the natural C-1027.
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Affiliation(s)
- Toyonobu Usuki
- Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan
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Usuki T, Inoue M, Akiyama K, Hirama M. Spin-trapping Study of DNA Cleavage Induced by Enediyne C-1027 Chromophore. CHEM LETT 2002. [DOI: 10.1246/cl.2002.1148] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Urbaniak MD, Muskett FW, Finucane MD, Caddick S, Woolfson DN. Solution Structure of a Novel Chromoprotein Derived from Apo-Neocarzinostatin and a Synthetic Chromophore. Biochemistry 2002; 41:11731-9. [PMID: 12269815 DOI: 10.1021/bi0262146] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The natural complex Neocarzinostatin comprises a labile chromophore noncovalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apoprotein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a location similar to that observed for the chromophore in the natural Neocarzinostatin complex, but with a distinct orientation. These results provide structural evidence that the apoprotein can readily accommodate small druglike entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex described by others, together with the results reported here, suggests potential applications for small molecule binding by apo-Neocarzinostatin.
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Affiliation(s)
- Michael D Urbaniak
- Centre for Biomolecular Design and Drug Development, CPES, University of Sussex, Falmer, Lewes Road, Brighton BN1 9QJ, U.K
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Sasaki T, Inoue M, Hirama M. Synthetic studies toward C-1027 chromophore: construction of a highly unsaturated macrocycle. Tetrahedron Lett 2001. [DOI: 10.1016/s0040-4039(01)00959-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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