A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

Protein Eng Des Sel. 2007 Nov;20(11):569-76. doi: 10.1093/protein/gzm061. Epub 2007 Nov 3.

Abstract

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Albumins / metabolism*
  • Animals
  • Antibodies / genetics
  • Antibodies / immunology*
  • Antibodies / metabolism*
  • Antigens / immunology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Humans
  • Interleukin-2 / metabolism
  • Lymphocyte Activation / drug effects
  • Lymphocyte Activation / immunology
  • Mice
  • Models, Molecular
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / metabolism*
  • Recombinant Fusion Proteins / pharmacokinetics
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism

Substances

  • Albumins
  • Antibodies
  • Antigens
  • Bacterial Proteins
  • IgG Fc-binding protein, Streptococcus
  • Interleukin-2
  • Recombinant Fusion Proteins