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

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.