Abstract
The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.
Highlights
Whole antibodies, especially chimeric, humanized or fully human IgG molecules, exhibit a long circulation time in the human body that can reach a half-life of 27 days [1, 2]
B, size exclusion chromatography of unmodified and N-glycosylated single-chain diabody (scDb) molecules. c, ELISA of binding of unmodified and N-glycosylated scDb variants to immobilized CEA (n ϭ 2). d, flow cytometry analysis of binding of unmodified and N-glycosylated scDb variants to CEA-expressing tumor cells (LS174T), CD3-positive PBMCs, or HEK293 cells included as negative control
Yields were similar to that obtained for the unmodified scDb indicating that the modifications do not interfere with translation and secretion into the cell culture supernatant
Summary
Materials—Horseradish peroxidase-conjugated anti-His tag antibody was purchased from Santa Cruz Biotechnology, unconjugated anti-His tag antibody from Dianova (Hamburg, Germany) and anti-mouse IgG-FITC or PE-conjugated antibody as well as goat anti-rabbit IgG-FITC-conjugated antibody from Sigma (Taufkirchen, Germany). Protein Expression and Purification—Plasmid DNA encoding the respective recombinant antibody was transfected with LipofectamineTM 2000 (Invitrogen) into HEK293 cells. Flow Cytometry—5 ϫ 105 cells/well were incubated with recombinant antibodies (10 g/ml) for 2 h at 4 °C. Antibody or rabbit antiserum followed by washing and 30 min of incubation with PE-labeled anti-mouse IgG or FITC-conjugated goat anti-rabbit antibody. An aliquot of 25 l of methyl iodide was added to the reaction mixture followed by incubation for another 30 min at room temperature. The day, the supernatant was removed, and 100 l of recombinant antibody in RPMI, 10% FBS added. Antihuman IL-2 antibodies as well as the standard of recombinant human IL-2 was provided by DuoSet IL-2 ELISA Development System kit (R&D Systems, Nordenstadt, Germany), and the assay was performed following the manufacturer’s protocol.
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