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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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Related in: MedlinePlus

Purification and binding affinity of the 3E8-mEndo fusion protein(A) Schematic diagram of the anti-3E8 humanized antibody and the 3E8-mEndo fusion protein. (B) Expression of anti-3E8 antibody and 3E8-mEndo. Secreted 3E8 and 3E8-mEndo were analyzed under reducing and non-reducing conditions. (C) HUVECs and LS174T cells were treated with 3E8, 3E8-mEndo, and mEndo. The binding affinities of 3E8 and 3E8-mEndo were detected with the anti-3E8 antibody, and mEndo was detected by anti-mouse endostatin. Experiments were repeated three times. Isotype control, human IgG, filled with red.
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Figure 1: Purification and binding affinity of the 3E8-mEndo fusion protein(A) Schematic diagram of the anti-3E8 humanized antibody and the 3E8-mEndo fusion protein. (B) Expression of anti-3E8 antibody and 3E8-mEndo. Secreted 3E8 and 3E8-mEndo were analyzed under reducing and non-reducing conditions. (C) HUVECs and LS174T cells were treated with 3E8, 3E8-mEndo, and mEndo. The binding affinities of 3E8 and 3E8-mEndo were detected with the anti-3E8 antibody, and mEndo was detected by anti-mouse endostatin. Experiments were repeated three times. Isotype control, human IgG, filled with red.

Mentions: We constructed and stably transfected an expression vector for the 3E8-mEndo fusion protein, comprising mouse endostatin (mEndo) fused to the C-terminal end of 3E8 antibody with a 17 amino acid-linker (Figure 1A), into dihydrofolate reductase-deficient CHO-DG44 cells. Due to the presence of an endogenous Igκ leader sequence, the expressed 3E8-mEndo fusion protein was secreted. Secreted 3E8 and 3E8-mEndo fusion proteins were purified from culture supernatants using protein A-sepharose affinity column chromatography. Purified 3E8 and 3E8-mEndo proteins were analyzed by SDS-PAGE under non-reducing or reducing conditions. Purified 3E8-mEndo migrated as a 190 kDa band under non-reducing conditions, indicating that it is composed of 3E8 (150 kDa) and two endostatin molecules (40 kDa) (Figure 1B). Western blot analyses using polyclonal anti-human IgG showed an approximately 25 kDa immunoreactive band corresponding to light chains of both 3E8 and 3E8-mEndo, as well as two bands with molecular masses of ~50 kDa and ~70 kDa, corresponding to heavy chain and heavy chain plus endostatin of 3E8 and 3E8-mEndo, respectively (Figure 1B). In contrast, immunoblotting with the anti-mEndostatin antibody only detects the 70 kDa polypeptide comprising heavy chain plus endostatin of the 3E8-mEndo fusion protein (Figure 1B).


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)

Purification and binding affinity of the 3E8-mEndo fusion protein(A) Schematic diagram of the anti-3E8 humanized antibody and the 3E8-mEndo fusion protein. (B) Expression of anti-3E8 antibody and 3E8-mEndo. Secreted 3E8 and 3E8-mEndo were analyzed under reducing and non-reducing conditions. (C) HUVECs and LS174T cells were treated with 3E8, 3E8-mEndo, and mEndo. The binding affinities of 3E8 and 3E8-mEndo were detected with the anti-3E8 antibody, and mEndo was detected by anti-mouse endostatin. Experiments were repeated three times. Isotype control, human IgG, filled with red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Purification and binding affinity of the 3E8-mEndo fusion protein(A) Schematic diagram of the anti-3E8 humanized antibody and the 3E8-mEndo fusion protein. (B) Expression of anti-3E8 antibody and 3E8-mEndo. Secreted 3E8 and 3E8-mEndo were analyzed under reducing and non-reducing conditions. (C) HUVECs and LS174T cells were treated with 3E8, 3E8-mEndo, and mEndo. The binding affinities of 3E8 and 3E8-mEndo were detected with the anti-3E8 antibody, and mEndo was detected by anti-mouse endostatin. Experiments were repeated three times. Isotype control, human IgG, filled with red.
Mentions: We constructed and stably transfected an expression vector for the 3E8-mEndo fusion protein, comprising mouse endostatin (mEndo) fused to the C-terminal end of 3E8 antibody with a 17 amino acid-linker (Figure 1A), into dihydrofolate reductase-deficient CHO-DG44 cells. Due to the presence of an endogenous Igκ leader sequence, the expressed 3E8-mEndo fusion protein was secreted. Secreted 3E8 and 3E8-mEndo fusion proteins were purified from culture supernatants using protein A-sepharose affinity column chromatography. Purified 3E8 and 3E8-mEndo proteins were analyzed by SDS-PAGE under non-reducing or reducing conditions. Purified 3E8-mEndo migrated as a 190 kDa band under non-reducing conditions, indicating that it is composed of 3E8 (150 kDa) and two endostatin molecules (40 kDa) (Figure 1B). Western blot analyses using polyclonal anti-human IgG showed an approximately 25 kDa immunoreactive band corresponding to light chains of both 3E8 and 3E8-mEndo, as well as two bands with molecular masses of ~50 kDa and ~70 kDa, corresponding to heavy chain and heavy chain plus endostatin of 3E8 and 3E8-mEndo, respectively (Figure 1B). In contrast, immunoblotting with the anti-mEndostatin antibody only detects the 70 kDa polypeptide comprising heavy chain plus endostatin of the 3E8-mEndo fusion protein (Figure 1B).

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