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Extension of the in vivo half-life of endostatin and its improved anti-tumor activities upon fusion to a humanized antibody against tumor-associated glycoprotein 72 in a mouse model of human colorectal carcinoma.

Lee SH, Jeung IC, Park TW, Lee K, Lee DG, Cho YL, Lee TS, Na HJ, Park YJ, Lee HG, Jeong MS, Bae KH, Lee SC, Lee HJ, Kwon YG, Hong HJ, Kim JS, Min JK - Oncotarget (2015)

Bottom Line: Endostatin is an endogenous angiogenesis inhibitor that exhibits potential anti-tumor efficacy in various preclinical animal models.However, its relatively short in vivo half-life and the long-term, frequent administration of high doses limit its widespread clinical use.Moreover, in mice treated with 3E8-mEndo, we observed a markedly prolonged serum half-life and significantly inhibited tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

ABSTRACT
Endostatin is an endogenous angiogenesis inhibitor that exhibits potential anti-tumor efficacy in various preclinical animal models. However, its relatively short in vivo half-life and the long-term, frequent administration of high doses limit its widespread clinical use. In this study, we evaluated whether a fusion protein of murine endostatin (mEndo) to a humanized antibody against tumor-associated glycoprotein 72 (TAG-72), which is highly expressed in several human tumor tissues including colon cancer, can extend the serum half-life and improve the anti-tumor efficacy of endostatin by targeted delivery to the tumor mass. The fusion protein (3E8-mEndo) and mEndo showed improved anti-angiogenic activity in vitro and in vivo, predominantly by interfering with pro-angiogenic signaling triggered by vascular endothelial growth factor (VEGF). Moreover, in mice treated with 3E8-mEndo, we observed a markedly prolonged serum half-life and significantly inhibited tumor growth. The improved anti-tumor activity of 3E8-mEndo can be partially explained by increased local concentration in the tumor mass due to targeted delivery of 3E8-mEndo to implanted colon tumors. Collectively, our data clearly indicate that tumor-targeting antibody fusions to endostatin are a powerful strategy that improves the poor pharmacokinetic profile and anti-tumor efficacy of endostatin.

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The effects of the 3E8-mEndo fusion protein on VEGF-induced endothelial cell proliferation, migration, and capillary-like tube formation(A) HUVECs were pretreated with 3E8 antibody, mEndo, and 3E8-mEndo for 30 min, and then stimulated with 10 ng/ml VEGF165. After incubation for 48 h, live cells were counted under a microscope. Each group was assayed in triplicate. (B) HUVECs were assayed for chemotaxis toward 10 ng/ml VEGF in the continued presence or absence of the fusion protein and mEndo. Cells that migrated to the bottom of transwell membranes were stained with hematoxylin and eosin and quantified under a microscope. Quantification of five independent assays is shown in the graphs (A and B). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (C) HUVECs were resuspended in endothelial cell growth media and treated as indicated before plating onto Matrigel-coated plates. After incubation for 20 h, tube formation was observed using an inverted microscope. Each group was assayed in triplicate and experiments were repeated three times. Scale bar: 10 μm.
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Figure 2: The effects of the 3E8-mEndo fusion protein on VEGF-induced endothelial cell proliferation, migration, and capillary-like tube formation(A) HUVECs were pretreated with 3E8 antibody, mEndo, and 3E8-mEndo for 30 min, and then stimulated with 10 ng/ml VEGF165. After incubation for 48 h, live cells were counted under a microscope. Each group was assayed in triplicate. (B) HUVECs were assayed for chemotaxis toward 10 ng/ml VEGF in the continued presence or absence of the fusion protein and mEndo. Cells that migrated to the bottom of transwell membranes were stained with hematoxylin and eosin and quantified under a microscope. Quantification of five independent assays is shown in the graphs (A and B). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (C) HUVECs were resuspended in endothelial cell growth media and treated as indicated before plating onto Matrigel-coated plates. After incubation for 20 h, tube formation was observed using an inverted microscope. Each group was assayed in triplicate and experiments were repeated three times. Scale bar: 10 μm.

Mentions: To determine whether the fusion of endostatin to 3E8 affects the anti-angiogenic activities of endostatin, we examined the effects of 3E8-mEndo on endothelial cell proliferation, migration, and capillary-like tube formation in response to VEGF. VEGF stimulation significantly induced HUVEC proliferation and migration (Figure 2A and 2B). However, pre-treating cells with mEndo or 3E8-mEndo significantly inhibited VEGF-induced proliferation and migration in HUVECs. The anti-angiogenic activity of the 3E8-mEndo fusion protein appears to be higher than mEndo because cells treated with 10 μg/ml of 3E8-mEndo more potently inhibited VEGF-induced proliferation and migration compared to cells treated with an equal concentration of mEndo. Additionally, the 3E8 antibody did not affect cell proliferation and migration in HUVECs in the presence or absence of VEGF. Moreover, cells treated with 3E8 or 3E8-mEndo without VEGF stimulation did not exhibit any toxicity. Next, we examined the effects of the 3E8-mEndo fusion protein on capillary-like tube formation in vitro. HUVECs were plated on growth factor-reduced Matrigel, and then pretreated with mEndo, 3E8, and 3E8-mEndo in the presence or absence of VEGF (10 ng/ml). Tube formation was determined by the presence of cells organized as closed polygons. mEndo treatment significantly reduced VEGF-induced tube formation, whereas the 3E8 antibody showed no significant effects. Interestingly, cells treated with 3E8-mEndo showed substantially inhibited VEGF-induced tube formation in a dose-dependent manner, which was dramatically higher than cells treated with mEndo.


Extension of the in vivo half-life of endostatin and its improved anti-tumor activities upon fusion to a humanized antibody against tumor-associated glycoprotein 72 in a mouse model of human colorectal carcinoma.

Lee SH, Jeung IC, Park TW, Lee K, Lee DG, Cho YL, Lee TS, Na HJ, Park YJ, Lee HG, Jeong MS, Bae KH, Lee SC, Lee HJ, Kwon YG, Hong HJ, Kim JS, Min JK - Oncotarget (2015)

The effects of the 3E8-mEndo fusion protein on VEGF-induced endothelial cell proliferation, migration, and capillary-like tube formation(A) HUVECs were pretreated with 3E8 antibody, mEndo, and 3E8-mEndo for 30 min, and then stimulated with 10 ng/ml VEGF165. After incubation for 48 h, live cells were counted under a microscope. Each group was assayed in triplicate. (B) HUVECs were assayed for chemotaxis toward 10 ng/ml VEGF in the continued presence or absence of the fusion protein and mEndo. Cells that migrated to the bottom of transwell membranes were stained with hematoxylin and eosin and quantified under a microscope. Quantification of five independent assays is shown in the graphs (A and B). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (C) HUVECs were resuspended in endothelial cell growth media and treated as indicated before plating onto Matrigel-coated plates. After incubation for 20 h, tube formation was observed using an inverted microscope. Each group was assayed in triplicate and experiments were repeated three times. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466677&req=5

Figure 2: The effects of the 3E8-mEndo fusion protein on VEGF-induced endothelial cell proliferation, migration, and capillary-like tube formation(A) HUVECs were pretreated with 3E8 antibody, mEndo, and 3E8-mEndo for 30 min, and then stimulated with 10 ng/ml VEGF165. After incubation for 48 h, live cells were counted under a microscope. Each group was assayed in triplicate. (B) HUVECs were assayed for chemotaxis toward 10 ng/ml VEGF in the continued presence or absence of the fusion protein and mEndo. Cells that migrated to the bottom of transwell membranes were stained with hematoxylin and eosin and quantified under a microscope. Quantification of five independent assays is shown in the graphs (A and B). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (C) HUVECs were resuspended in endothelial cell growth media and treated as indicated before plating onto Matrigel-coated plates. After incubation for 20 h, tube formation was observed using an inverted microscope. Each group was assayed in triplicate and experiments were repeated three times. Scale bar: 10 μm.
Mentions: To determine whether the fusion of endostatin to 3E8 affects the anti-angiogenic activities of endostatin, we examined the effects of 3E8-mEndo on endothelial cell proliferation, migration, and capillary-like tube formation in response to VEGF. VEGF stimulation significantly induced HUVEC proliferation and migration (Figure 2A and 2B). However, pre-treating cells with mEndo or 3E8-mEndo significantly inhibited VEGF-induced proliferation and migration in HUVECs. The anti-angiogenic activity of the 3E8-mEndo fusion protein appears to be higher than mEndo because cells treated with 10 μg/ml of 3E8-mEndo more potently inhibited VEGF-induced proliferation and migration compared to cells treated with an equal concentration of mEndo. Additionally, the 3E8 antibody did not affect cell proliferation and migration in HUVECs in the presence or absence of VEGF. Moreover, cells treated with 3E8 or 3E8-mEndo without VEGF stimulation did not exhibit any toxicity. Next, we examined the effects of the 3E8-mEndo fusion protein on capillary-like tube formation in vitro. HUVECs were plated on growth factor-reduced Matrigel, and then pretreated with mEndo, 3E8, and 3E8-mEndo in the presence or absence of VEGF (10 ng/ml). Tube formation was determined by the presence of cells organized as closed polygons. mEndo treatment significantly reduced VEGF-induced tube formation, whereas the 3E8 antibody showed no significant effects. Interestingly, cells treated with 3E8-mEndo showed substantially inhibited VEGF-induced tube formation in a dose-dependent manner, which was dramatically higher than cells treated with mEndo.

Bottom Line: Endostatin is an endogenous angiogenesis inhibitor that exhibits potential anti-tumor efficacy in various preclinical animal models.However, its relatively short in vivo half-life and the long-term, frequent administration of high doses limit its widespread clinical use.Moreover, in mice treated with 3E8-mEndo, we observed a markedly prolonged serum half-life and significantly inhibited tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

ABSTRACT
Endostatin is an endogenous angiogenesis inhibitor that exhibits potential anti-tumor efficacy in various preclinical animal models. However, its relatively short in vivo half-life and the long-term, frequent administration of high doses limit its widespread clinical use. In this study, we evaluated whether a fusion protein of murine endostatin (mEndo) to a humanized antibody against tumor-associated glycoprotein 72 (TAG-72), which is highly expressed in several human tumor tissues including colon cancer, can extend the serum half-life and improve the anti-tumor efficacy of endostatin by targeted delivery to the tumor mass. The fusion protein (3E8-mEndo) and mEndo showed improved anti-angiogenic activity in vitro and in vivo, predominantly by interfering with pro-angiogenic signaling triggered by vascular endothelial growth factor (VEGF). Moreover, in mice treated with 3E8-mEndo, we observed a markedly prolonged serum half-life and significantly inhibited tumor growth. The improved anti-tumor activity of 3E8-mEndo can be partially explained by increased local concentration in the tumor mass due to targeted delivery of 3E8-mEndo to implanted colon tumors. Collectively, our data clearly indicate that tumor-targeting antibody fusions to endostatin are a powerful strategy that improves the poor pharmacokinetic profile and anti-tumor efficacy of endostatin.

Show MeSH
Related in: MedlinePlus