The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Victor C. Yang - One of the best experts on this subject based on the ideXlab platform.
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A self-assembly and stimuli-responsive fusion Gelonin delivery system for tumor treatment
Journal of Industrial and Engineering Chemistry, 2020Co-Authors: Quan Liu, Meong Cheol Shin, Jingwen Zhao, Lu Zhang, Shuping Xie, Baoyan Pan, Victor C. YangAbstract:Abstract Ribosome-inactivating proteins (RIPs) are potent protein toxins for cancer therapy, and they have strong ability to inhibit protein synthesis and induce cell death via inactivation of ribosomes in eukaryotic cells. However, the delivery of RIPs has been a challenging task due to their large molecular weight and lack of targeting property. Low molecular weight protamine (LMWP), a transmembrane peptide, has been proved to effectively promote transmembrane transportation, whereas the enzyme-activatable system can enhance the specificity by enhancing the tumor drug concentration through enzymatic reaction. We herein constructed a self-assembly and stimuli-responsive fusion Gelonin delivery system. Gelonin, a typical RIP protein, was assembled with nickel ferrite nanoparticles by self-assembling between hexa-histidine tag (His-tagged) and nickel ions. Both in vitro and in vivo results indicated that the magnetic nanoparticle carriers and the applied linkers did not damage the pharmaceutical effect of Gelonin, and the whole drug delivery system showed good biocompatibility, sensitive selectivity, and significantly enhanced cytotoxic activity. This in turn presented theranostic nanoparticles as efficient delivery vehicle for clinical use.
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Tandem-multimeric F3-Gelonin fusion toxins for enhanced anti-cancer activity for prostate cancer treatment.
International journal of pharmaceutics, 2017Co-Authors: Meong Cheol Shin, Yongzhuo Huang, Kyoung Ah Min, Heesun Cheong, Cheol Moon, Victor C. YangAbstract:Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as Gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, Gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered Gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-Gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified Gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with Gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-Gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-Gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified Gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs.
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recombinant tat Gelonin fusion toxin synthesis and characterization of heparin protamine regulated cell transduction
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein toxins, such as Gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated Gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-Gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant Gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
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combination of antibody targeting and ptd mediated intracellular toxin delivery for colorectal cancer therapy
Journal of Controlled Release, 2014Co-Authors: Meong Cheol Shin, Jian Zhang, Kyoung Ah Min, Cheol Moon, Kyuri Lee, Joseph P Balthasar, Victor C. YangAbstract:The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin Gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-Gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified Gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-Gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-Gelonin to the tumor target was observed, when compared with administration of TAT-Gelonin alone. More importantly, efficacy studies also revealed that only the TAT-Gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing Gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.
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Recombinant TAT-Gelonin Fusion Toxin: Synthesis and Characterization of Heparin/Protamine-Regulated Cell Transduction
Journal of biomedical materials research. Part A, 2014Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein toxins, such as Gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated Gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-Gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant Gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
Meong Cheol Shin - One of the best experts on this subject based on the ideXlab platform.
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A self-assembly and stimuli-responsive fusion Gelonin delivery system for tumor treatment
Journal of Industrial and Engineering Chemistry, 2020Co-Authors: Quan Liu, Meong Cheol Shin, Jingwen Zhao, Lu Zhang, Shuping Xie, Baoyan Pan, Victor C. YangAbstract:Abstract Ribosome-inactivating proteins (RIPs) are potent protein toxins for cancer therapy, and they have strong ability to inhibit protein synthesis and induce cell death via inactivation of ribosomes in eukaryotic cells. However, the delivery of RIPs has been a challenging task due to their large molecular weight and lack of targeting property. Low molecular weight protamine (LMWP), a transmembrane peptide, has been proved to effectively promote transmembrane transportation, whereas the enzyme-activatable system can enhance the specificity by enhancing the tumor drug concentration through enzymatic reaction. We herein constructed a self-assembly and stimuli-responsive fusion Gelonin delivery system. Gelonin, a typical RIP protein, was assembled with nickel ferrite nanoparticles by self-assembling between hexa-histidine tag (His-tagged) and nickel ions. Both in vitro and in vivo results indicated that the magnetic nanoparticle carriers and the applied linkers did not damage the pharmaceutical effect of Gelonin, and the whole drug delivery system showed good biocompatibility, sensitive selectivity, and significantly enhanced cytotoxic activity. This in turn presented theranostic nanoparticles as efficient delivery vehicle for clinical use.
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Fusion of Gelonin and anti-insulin-like growth factor-1 receptor (IGF-1R) affibody for enhanced brain cancer therapy.
Archives of pharmacal research, 2017Co-Authors: Songhee Ham, Kyoung Ah Min, Jae Wook Yang, Meong Cheol ShinAbstract:Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as Gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered Gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody (“IAFF”) via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant Gelonin-anti-IGF-1R affibody (rGel-IAFF), Gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and Gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components’ functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of Gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than Gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC50 values (avg. IC50: 180 and 720 nM, respectively) than Gelonin (avg. IC50: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of Gelonin with IAFF could provide an effective way to enhance the anti-tumor activity, while reducing the associated toxicity of Gelonin.
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Molecular tumor targeting of Gelonin by fusion with F3 peptide.
Acta pharmacologica Sinica, 2017Co-Authors: Songhee Ham, Kyoung Ah Min, Meong Cheol ShinAbstract:Therapeutically potent macromolecular drugs have shown great promise for overcoming the limitations of small-molecule anti-cancer drugs. But tumor cell-selective intracellular delivery of the macromolecules remains a major hurdle for their successful clinical application. To overcome this challenge, we engineered a novel genetic fusion protein (F3-Gel) that composed of F3 peptide, a tumor-homing peptide, and Gelonin, a plant-derived ribosome-inactivating protein (RIP), and then evaluated its anti-cancer activity in vitro and in vivo. The F3-Gel-encoding gene was synthesized by genetic recombination, and F3-Gel was successfully expressed in E coli. The anti-cancer activity of the produced F3-Gel was evaluated by various in vitro assays, which revealed that F3-Gel maintained equipotent protein synthesis inhibition activity (IC50=11 pmol/L) as unmodified Gelonin (IC50=10 pmol/L). Furthermore, F3-Gel displayed enhanced cellular uptake into cancer cells (U87 MG, HeLa, LnCaP and 9L) than noncancerous cells (293 HEK and SVGp12). Compared with Gelonin, F3-Gel exerted significantly higher cytotoxicity against these cancer cells. F3-Gel displayed significantly greater inhibition of protein translation in U87 MG cells: F3-Gel (0.5 μmol/L) was able to reduce the protein level to less than 50%, while Gelonin (1 μmol/L) did not affect the intracellular protein level. In a U87 MG xenograft tumor-bearing mouse model, F3-Gel was accumulated in the tumor site at much higher levels and maintained for a prolonged time compared with Gelonin. Administration of F3-Gel (0.5, 0.75 mol/kg, iv) caused 36% and 66%, respectively, inhibition of tumor growth in U87 MG xenograft mice, suggesting that it is a promising candidate drug for cancer treatment. Furthermore, this study demonstrates that fusion of F3 peptide to a potent macromolecule could provides an effective method for targeting tumors and eventually could improve their druggability.
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Tandem-multimeric F3-Gelonin fusion toxins for enhanced anti-cancer activity for prostate cancer treatment.
International journal of pharmaceutics, 2017Co-Authors: Meong Cheol Shin, Yongzhuo Huang, Kyoung Ah Min, Heesun Cheong, Cheol Moon, Victor C. YangAbstract:Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as Gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, Gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered Gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-Gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified Gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with Gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-Gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-Gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified Gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs.
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recombinant tat Gelonin fusion toxin synthesis and characterization of heparin protamine regulated cell transduction
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein toxins, such as Gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated Gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-Gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant Gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
Christopher M Pirie - One of the best experts on this subject based on the ideXlab platform.
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Conflict of Interest
2016Co-Authors: Christopher M Pirie, Dane K Wittrup, David V. LiuAbstract:Targeted cytolysins synergistically potentiate cytoplasmic delivery of Gelonin immunotoxi
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Large Molecule Therapeutics Targeted
2016Co-Authors: Cytolysins Synergistically Potentiate Cytoplasmic, Christopher M Pirie, Delivery Of Gelonin Immunotoxin, David V. Liu, Dane K WittrupAbstract:Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an inde-pendently targeted potent Gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin Gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized Gelonin molecules required to induce apoptosis is reduced from approximately 5 106 to less than 103. Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA. Mol Cancer Ther; 12(9); 1774–82. 2013 AACR
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Targeted cytolysins synergistically potentiate cytoplasmic delivery of Gelonin immunotoxin
Molecular cancer therapeutics, 2013Co-Authors: Christopher M Pirie, David Liu, K. Dane WittrupAbstract:Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an independently targeted potent Gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin Gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized Gelonin molecules required to induce apoptosis is reduced from approximately 5 × 10(6) to less than 10(3). Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA.
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convergent potency of internalized Gelonin immunotoxins across varied cell lines antigens and targeting moieties
Journal of Biological Chemistry, 2011Co-Authors: Christopher M Pirie, Benjamin J Hackel, Michael G Rosenblum, Dane K WittrupAbstract:Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel Gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with Gelonin (IC(50) ∼ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of Gelonin (via pinocytosis) and Gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ∼ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of Gelonin-based immunotoxins.
Dane K Wittrup - One of the best experts on this subject based on the ideXlab platform.
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Conflict of Interest
2016Co-Authors: Christopher M Pirie, Dane K Wittrup, David V. LiuAbstract:Targeted cytolysins synergistically potentiate cytoplasmic delivery of Gelonin immunotoxi
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Large Molecule Therapeutics Targeted
2016Co-Authors: Cytolysins Synergistically Potentiate Cytoplasmic, Christopher M Pirie, Delivery Of Gelonin Immunotoxin, David V. Liu, Dane K WittrupAbstract:Targeted endocytic uptake is a first step toward tissue-specific cytoplasmic macromolecular delivery; however, inefficient escape from the endolysosomal compartment makes this generally impractical at present. We report here a targeted cytolysin approach that dramatically potentiates endosomal release of an inde-pendently targeted potent Gelonin immunotoxin. Fibronectin domains engineered for affinity to EGF receptor or carcinoembryonic antigen were fused to the plant toxin Gelonin or bacterial pore-forming cytolysins. These fusion proteins display synergistic activity in both antigen-specific cytotoxicity in vitro, enhancing potency by several orders of magnitude, and in tumor growth inhibition in vivo. In addition, the number of internalized Gelonin molecules required to induce apoptosis is reduced from approximately 5 106 to less than 103. Targeted potentiation shows promise for enhancing cytoplasmic delivery of other macromolecular payloads such as DNA, siRNA, and miRNA. Mol Cancer Ther; 12(9); 1774–82. 2013 AACR
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Synergistic Antitumor Activity from Two-Stage Delivery of Targeted Toxins and Endosome-Disrupting Nanoparticles
2015Co-Authors: Nicole Yang, Dane K Wittrup, Darrell J. IrvineAbstract:Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted Gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound Gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of Gelonin to be focused on tumors with significantly reduced potential for off-target toxicity
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convergent potency of internalized Gelonin immunotoxins across varied cell lines antigens and targeting moieties
Journal of Biological Chemistry, 2011Co-Authors: Christopher M Pirie, Benjamin J Hackel, Michael G Rosenblum, Dane K WittrupAbstract:Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel Gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with Gelonin (IC(50) ∼ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of Gelonin (via pinocytosis) and Gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ∼ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of Gelonin-based immunotoxins.
Kyoung Ah Min - One of the best experts on this subject based on the ideXlab platform.
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Fusion of Gelonin and anti-insulin-like growth factor-1 receptor (IGF-1R) affibody for enhanced brain cancer therapy.
Archives of pharmacal research, 2017Co-Authors: Songhee Ham, Kyoung Ah Min, Jae Wook Yang, Meong Cheol ShinAbstract:Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as Gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered Gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody (“IAFF”) via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant Gelonin-anti-IGF-1R affibody (rGel-IAFF), Gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and Gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components’ functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of Gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than Gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC50 values (avg. IC50: 180 and 720 nM, respectively) than Gelonin (avg. IC50: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of Gelonin with IAFF could provide an effective way to enhance the anti-tumor activity, while reducing the associated toxicity of Gelonin.
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Molecular tumor targeting of Gelonin by fusion with F3 peptide.
Acta pharmacologica Sinica, 2017Co-Authors: Songhee Ham, Kyoung Ah Min, Meong Cheol ShinAbstract:Therapeutically potent macromolecular drugs have shown great promise for overcoming the limitations of small-molecule anti-cancer drugs. But tumor cell-selective intracellular delivery of the macromolecules remains a major hurdle for their successful clinical application. To overcome this challenge, we engineered a novel genetic fusion protein (F3-Gel) that composed of F3 peptide, a tumor-homing peptide, and Gelonin, a plant-derived ribosome-inactivating protein (RIP), and then evaluated its anti-cancer activity in vitro and in vivo. The F3-Gel-encoding gene was synthesized by genetic recombination, and F3-Gel was successfully expressed in E coli. The anti-cancer activity of the produced F3-Gel was evaluated by various in vitro assays, which revealed that F3-Gel maintained equipotent protein synthesis inhibition activity (IC50=11 pmol/L) as unmodified Gelonin (IC50=10 pmol/L). Furthermore, F3-Gel displayed enhanced cellular uptake into cancer cells (U87 MG, HeLa, LnCaP and 9L) than noncancerous cells (293 HEK and SVGp12). Compared with Gelonin, F3-Gel exerted significantly higher cytotoxicity against these cancer cells. F3-Gel displayed significantly greater inhibition of protein translation in U87 MG cells: F3-Gel (0.5 μmol/L) was able to reduce the protein level to less than 50%, while Gelonin (1 μmol/L) did not affect the intracellular protein level. In a U87 MG xenograft tumor-bearing mouse model, F3-Gel was accumulated in the tumor site at much higher levels and maintained for a prolonged time compared with Gelonin. Administration of F3-Gel (0.5, 0.75 mol/kg, iv) caused 36% and 66%, respectively, inhibition of tumor growth in U87 MG xenograft mice, suggesting that it is a promising candidate drug for cancer treatment. Furthermore, this study demonstrates that fusion of F3 peptide to a potent macromolecule could provides an effective method for targeting tumors and eventually could improve their druggability.
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Tandem-multimeric F3-Gelonin fusion toxins for enhanced anti-cancer activity for prostate cancer treatment.
International journal of pharmaceutics, 2017Co-Authors: Meong Cheol Shin, Yongzhuo Huang, Kyoung Ah Min, Heesun Cheong, Cheol Moon, Victor C. YangAbstract:Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as Gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, Gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered Gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-Gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified Gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with Gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-Gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-Gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified Gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs.
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recombinant tat Gelonin fusion toxin synthesis and characterization of heparin protamine regulated cell transduction
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein toxins, such as Gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated Gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-Gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant Gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
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combination of antibody targeting and ptd mediated intracellular toxin delivery for colorectal cancer therapy
Journal of Controlled Release, 2014Co-Authors: Meong Cheol Shin, Jian Zhang, Kyoung Ah Min, Cheol Moon, Kyuri Lee, Joseph P Balthasar, Victor C. YangAbstract:The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin Gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-Gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified Gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-Gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-Gelonin to the tumor target was observed, when compared with administration of TAT-Gelonin alone. More importantly, efficacy studies also revealed that only the TAT-Gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing Gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.