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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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3E8-mEndo significantly inhibits VEGF receptor-2 activation(A) 3E8-mEndo and mEndo inhibit VEGF-induced VEGFR-2 phosphorylation. HUVECs were pretreated with 3E8-mEndo, mEndo, and 3E8 (10 μg/ml) for 30 min, and then stimulated with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting using anti-pVEGFR2 at Tyr1175 and anti-VEGFR2 antibodies. Quantification of VEGFR2 phosphorylation from five independent experiments is shown in the graphs. *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (B and C) Pretreatment of HUVECs with 3E8-mEndo and mEndo (10 ug/ml) for 30 min, followed by stimulation with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting. (D) The 3E8-mEndo fusion protein reduced VEGF-induced F-actin and focal adhesion formation. HUVECs were plated on coverslips coated with 20 μg/ml gelatin. After pretreatment with 3E8-mEndo and mEndo for 30 min, cells were stimulated with 10 ng/ml VEGF for 10 min. Immunostaining with the anti-mouse-paxillin antibody and TRITC-labeled phalloidin was performed on fixed cells. The size and number of focal adhesions were measured from the images (n = 15). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. Scale bar: 20 μm.
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Figure 4: 3E8-mEndo significantly inhibits VEGF receptor-2 activation(A) 3E8-mEndo and mEndo inhibit VEGF-induced VEGFR-2 phosphorylation. HUVECs were pretreated with 3E8-mEndo, mEndo, and 3E8 (10 μg/ml) for 30 min, and then stimulated with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting using anti-pVEGFR2 at Tyr1175 and anti-VEGFR2 antibodies. Quantification of VEGFR2 phosphorylation from five independent experiments is shown in the graphs. *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (B and C) Pretreatment of HUVECs with 3E8-mEndo and mEndo (10 ug/ml) for 30 min, followed by stimulation with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting. (D) The 3E8-mEndo fusion protein reduced VEGF-induced F-actin and focal adhesion formation. HUVECs were plated on coverslips coated with 20 μg/ml gelatin. After pretreatment with 3E8-mEndo and mEndo for 30 min, cells were stimulated with 10 ng/ml VEGF for 10 min. Immunostaining with the anti-mouse-paxillin antibody and TRITC-labeled phalloidin was performed on fixed cells. The size and number of focal adhesions were measured from the images (n = 15). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. Scale bar: 20 μm.

Mentions: Based on the findings demonstrating that endostatin suppresses VEGF-induced downstream signaling that is involved in endothelial cell proliferation and migration [14, 15] by directly binding to VEGFR-2 [3], we determined the effects of 3E8-mEndo on VEGF-induced VEGFR-2 signaling pathways. Stimulation of cells with VEGF (10 ng/ml) significantly increased VEGFR-2 phosphorylation at Tyr1175. However, pretreatment of cells with 3E8-mEndo or mEndo significantly reduced VEGF-induced phosphorylation of VEGFR-2 at Tyr1175 (Figure 4A). However, cells treated with the 3E8 antibody alone were unaffected. Activation of ERK1/2 and p38 MAPK, as well as the Src-FAK signaling cascade, induced by VEGFR-2 phosphorylation plays an important role in endothelial cells proliferation and migration [3, 16]. The 3E8-mEndo fusion protein significantly blocked VEGF-induced ERK1/2 and p38 MAPK phosphorylation in HUVECs (Figure 4B). Moreover, treatment of HUVECs with 3E8-mEndo significantly inhibited VEGF-induced FAK, Src, and paxillin phosphorylation (Figure 4C). Immunofluorescent staining with F-actin and paxillin showed that actin stress fiber formation and the number and size of focal adhesion in VEGF-stimulated HUVECs were markedly increased. However, cells treated with 3E8-mEndo or mEndo prior to VEGF stimulation showed highly impaired actin stress fiber and focal adhesion formation (Figure 4D). Similarly, LS174T cells treated with 3E8-mEndo or mEndo showed significant inhibition of bFGF-induced FGFR-2 activation and the downstream signaling pathways including Akt and Src phosphorylation (Supplementary Figure 3). Collectively, these results indicate that 3E8-mEndo can suppress VEGFR-2 and FGFR-2 signaling pathways in HUVECs and LS174T cells.


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)

3E8-mEndo significantly inhibits VEGF receptor-2 activation(A) 3E8-mEndo and mEndo inhibit VEGF-induced VEGFR-2 phosphorylation. HUVECs were pretreated with 3E8-mEndo, mEndo, and 3E8 (10 μg/ml) for 30 min, and then stimulated with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting using anti-pVEGFR2 at Tyr1175 and anti-VEGFR2 antibodies. Quantification of VEGFR2 phosphorylation from five independent experiments is shown in the graphs. *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (B and C) Pretreatment of HUVECs with 3E8-mEndo and mEndo (10 ug/ml) for 30 min, followed by stimulation with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting. (D) The 3E8-mEndo fusion protein reduced VEGF-induced F-actin and focal adhesion formation. HUVECs were plated on coverslips coated with 20 μg/ml gelatin. After pretreatment with 3E8-mEndo and mEndo for 30 min, cells were stimulated with 10 ng/ml VEGF for 10 min. Immunostaining with the anti-mouse-paxillin antibody and TRITC-labeled phalloidin was performed on fixed cells. The size and number of focal adhesions were measured from the images (n = 15). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: 3E8-mEndo significantly inhibits VEGF receptor-2 activation(A) 3E8-mEndo and mEndo inhibit VEGF-induced VEGFR-2 phosphorylation. HUVECs were pretreated with 3E8-mEndo, mEndo, and 3E8 (10 μg/ml) for 30 min, and then stimulated with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting using anti-pVEGFR2 at Tyr1175 and anti-VEGFR2 antibodies. Quantification of VEGFR2 phosphorylation from five independent experiments is shown in the graphs. *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. (B and C) Pretreatment of HUVECs with 3E8-mEndo and mEndo (10 ug/ml) for 30 min, followed by stimulation with 10 ng/ml VEGF for 10 min. Cell lysates were collected and analyzed by western blotting. (D) The 3E8-mEndo fusion protein reduced VEGF-induced F-actin and focal adhesion formation. HUVECs were plated on coverslips coated with 20 μg/ml gelatin. After pretreatment with 3E8-mEndo and mEndo for 30 min, cells were stimulated with 10 ng/ml VEGF for 10 min. Immunostaining with the anti-mouse-paxillin antibody and TRITC-labeled phalloidin was performed on fixed cells. The size and number of focal adhesions were measured from the images (n = 15). *p < 0.05; #p < 0.05 versus VEGF. Error bars indicate ± SEM. Scale bar: 20 μm.
Mentions: Based on the findings demonstrating that endostatin suppresses VEGF-induced downstream signaling that is involved in endothelial cell proliferation and migration [14, 15] by directly binding to VEGFR-2 [3], we determined the effects of 3E8-mEndo on VEGF-induced VEGFR-2 signaling pathways. Stimulation of cells with VEGF (10 ng/ml) significantly increased VEGFR-2 phosphorylation at Tyr1175. However, pretreatment of cells with 3E8-mEndo or mEndo significantly reduced VEGF-induced phosphorylation of VEGFR-2 at Tyr1175 (Figure 4A). However, cells treated with the 3E8 antibody alone were unaffected. Activation of ERK1/2 and p38 MAPK, as well as the Src-FAK signaling cascade, induced by VEGFR-2 phosphorylation plays an important role in endothelial cells proliferation and migration [3, 16]. The 3E8-mEndo fusion protein significantly blocked VEGF-induced ERK1/2 and p38 MAPK phosphorylation in HUVECs (Figure 4B). Moreover, treatment of HUVECs with 3E8-mEndo significantly inhibited VEGF-induced FAK, Src, and paxillin phosphorylation (Figure 4C). Immunofluorescent staining with F-actin and paxillin showed that actin stress fiber formation and the number and size of focal adhesion in VEGF-stimulated HUVECs were markedly increased. However, cells treated with 3E8-mEndo or mEndo prior to VEGF stimulation showed highly impaired actin stress fiber and focal adhesion formation (Figure 4D). Similarly, LS174T cells treated with 3E8-mEndo or mEndo showed significant inhibition of bFGF-induced FGFR-2 activation and the downstream signaling pathways including Akt and Src phosphorylation (Supplementary Figure 3). Collectively, these results indicate that 3E8-mEndo can suppress VEGFR-2 and FGFR-2 signaling pathways in HUVECs and LS174T cells.

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