Abstract
Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.
Highlights
All human serum albumin (HSA) fusion protein constructs, as well as scDbCEACD3 and the taFvCD3CEA were cloned as SfiI/EcoRI fragments into mammalian expression vector pSecTagA (Invitrogen, Karlsruhe, Germany)
ELISA—Binding properties of recombinant antibodies or antibody-HSA fusion proteins to CEA were analyzed by ELISA as following: 96-well plates were coated with carcinoembryonic antigen (300 ng/well) overnight at 4 °C
After addition of Peripheral Blood Mononuclear Cells (PBMC), the 96-well HSA fusion proteins were C-terminal endowed with a His plate was incubated for 22–24 h at 37 °C, 5% CO2
Summary
Diabody; AUC, area under the curve; CEA, carcinoembryonic antigen; HSA, human serum albumin; PBMC, peripheral blood mononuclear cell; PEG, polyethylene glycol; RSA, rat serum albumin; scFv, single chain Fv; scDb, single chain diabody; taFv, tandem scFv; IL, interleukin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography; FPLC, fast protein liquid chromatography; FAP, fibroblast activation protein; VH, heavy chain variable domain; VL, light chain variable domain. Recent studies have shown that this long serum half-life is due to a recycling process mediated by the neonatal Fc receptor (FcRn), similar to that observed for IgG molecules [23, 24] Taking advantage of these properties, human serum albumin (HSA) has been employed as macromolecular carrier for drug delivery or diagnostic purpose [19]. Three forms of recombinant bispecific antibody HSA fusion proteins based on single chain diabody, tandem scFv, or two different single chain Fv fragments were generated and produced in a mammalian expression system These novel bispecific antibody molecules showed specific binding to both antigens (CEA and CD3) and were able to retarget and activate effector cells in vitro to various extents. Compared with the parental antibodies, all bispecific albumin fusion proteins showed strong increase of the serum half-life in mice
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