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Improving the Oral Efficacy of Recombinant Granulocyte Colony-Stimulating Factor and Transferrin Fusion Protein by Spacer Optimization

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Abstract

Purpose

To improve the oral efficacy of the recombinant fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) by inserting a linker between the two protein domains.

Materials and Methods

Oligonucleotides encoding flexible and helix-forming peptides were inserted to the recombinant plasmids. The fusion protein without linker insertion was used for comparison. The G-CSF cell-proliferation and Tf receptor-binding activities of the fusion proteins were tested in NFS-60 cells and Caco-2 cells, respectively, and in vivo myelopoietic assay with both subcutaneous and oral administration was performed in BDF1 mice.

Results

All fusion proteins produced from transfected HEK293 cells were positive in Western-blotting assay with anti-G-CSF and anti-Tf antibodies. Among them, the fusion protein with a long helical (H4-2) linker showed the highest activity in NFS-60 cell proliferation assay, with an EC50 about ten-fold lower than that of the non-linker fusion protein. The fusion protein with H4-2 linker also showed a significantly higher myelopoietic effect when administered either subcutaneously or orally in BDF1 mice.

Conclusion

The insertion of a linker peptide, such as the helix linker H4-2, between G-CSF and Tf domains in the recombinant fusion protein can improve significantly both in vitro and in vivo myelopoietic activity over the non-linker fusion protein.

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Abbreviations

ANC:

absolute neutrophil count

G-CSF:

granulocyte colony stimulating factor

G-CSF-Tf:

recombinant fusion protein consisting of G-CSF and Tf

G-CSF-LE-Tf:

the fusion protein of G-CSF and Tf with a LE dipeptide linker

GS:

linker peptide with the amino acid sequence of (GGGGS)

H2:

linker peptide with the amino acid sequence of A(EAAAK)2A

H3:

linker peptide of A(EAAAK)3A

H4:

linker peptide of A(EAAAK)4A

H4-2:

linker peptide of A(EAAAK)4A

Tf:

transferrin

TfR:

transferrin receptor

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Acknowledgments

We thank Dr. James Ihle, St. Jude's Children's Research Center, Memphis, TN, for the generous gift of the NFS-60 cell line and Dr. David K. Ann for his helpful discussion. This work was supported in part by NIH grants GM063647 and HL064365.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, 90033, USA

    Yun Bai & Wei-Chiang Shen

  2. PSC 404B,1985 Zonal Avenue, Los Angeles, California, 90033, USA

    Wei-Chiang Shen

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  1. Yun Bai
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Correspondence to Wei-Chiang Shen.

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Bai, Y., Shen, WC. Improving the Oral Efficacy of Recombinant Granulocyte Colony-Stimulating Factor and Transferrin Fusion Protein by Spacer Optimization. Pharm Res 23, 2116–2121 (2006). https://doi.org/10.1007/s11095-006-9059-5

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  • DOI: https://doi.org/10.1007/s11095-006-9059-5

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