Structural Characterization of Human Recombinant and Bone-derived Bone Sialoprotein

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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 ϳ3-5-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.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). In contrast, antibody fragments, e.g. Fab fragments, or recombinant formats, such as single-chain Fv fragments (scFv) 3 or bispecific derivatives thereof (tandem scFv, diabodies, single-chain diabodies), are rapidly cleared from circulation (2-4). This is mainly due to the small size leading to rapid renal clearance and the lack of recycling processes mediated by the neonatal Fc receptor (FcRn). Thus, repeated injections or infusions are required to maintain a therapeutically effective dose in the body (5). Several strategies to improve pharmacokinetic properties and thus dosing and therapeutic efficacy of recombinant antibodies have been developed in recent years. These strategies can be divided into: 1) those based on reducing renal clearance by increasing the apparent size of the therapeutic molecule, and 2) those that in addition implement FcRn-mediated recycling processes (6 -8).PEGylation of proteins is a well-established strategy to improve pharmacokinetic properties by increasing the molecular mass and the hydrodynamic radius (9 -11). Several PEGylated proteins are in clinical use, e.g. PEGylated interferon ␣-2a (PegIntron, PEGASYS) and PEGylated granulocyte-colony stimulating factor (Neulasta), or are under clinical development, e.g. a PEGylated anti-TNF Fab fragment (certolizumab pegol) (9, 12) Furthermore, direct fusion to albumin or an albumin bin...

Section: Discussionmentioning

confidence: 99%

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Stork

Zettlitz

Müller

et al. 2008

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“…PCR-amplified cDNA was cloned in the pCRII vector (Invitrogen) and sequenced on both strands by cycle sequencing. NheI/XhoI restriction fragments of the pCRII-SMOC-1 plasmid were purified and cloned in the eukaryotic expression vector pCEP-Pu-His-Myc (41). Correct insertion of the construct in the pCEP-Pu-SMOC-1 was verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Characterization of SMOC-1, a Novel Modular Calcium-binding Protein in Basement Membranes

Vannahme¹,

Smyth²,

Miosge³

et al. 2002

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100

113

We have isolated the novel gene SMOC-1 that encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain. Recombinant expression in human cells showed that SMOC-1 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots, reverse transcriptase-PCR, and immunoblots revealed a widespread expression in many tissues. Immunofluorescence studies with an antiserum directed against recombinant human SMOC-1 demonstrated a basement membrane localization of the protein and additionally its presence in other extracellular matrices. Immunogold electron microscopy confirmed the localization of SMOC-1 within basement membranes in kidney and skeletal muscle as well as its expression in the zona pellucida surrounding the oocyte.

“…Studies by nuclear magnetic resonance, circular dichroism (CD), and small angle x-ray scattering have concluded that BSP has a flexible conformation with very little, if any, secondary structure (3)(4)(5)(6).…”

mentioning

confidence: 99%

Identification of the Type I Collagen-binding Domain of Bone Sialoprotein and Characterization of the Mechanism of Interaction

Tye

Hunter

Goldberg

2005

117

110

Bone sialoprotein (BSP) is an anionic phosphorylated glycoprotein that is expressed almost exclusively in mineralized tissues and has been shown to be a potent nucleator of hydroxyapatite formation. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and in the adhesion of bone cells to the mineralized matrix. Using a solid phase assay, we have investigated the interaction between BSP and collagen. Initial studies showed that raising the ionic strength, decreasing the pH below 7, or introducing divalent cations diminishes but does not abolish the binding of BSP to collagen, indicating that the interaction is only partly electrostatic in nature. Both boneextracted and recombinant (r)BSP exhibited similar binding affinities, indicating that post-translational modifications are not critical for binding. To identify the collagen-binding domain, recombinant peptides of BSP were studied. Peptide rBSP-(1-100) binds to type I collagen with an affinity similar to that of full-length rBSP, whereas peptides containing the sequences 99 -201 or 200 -301 do not bind. Further studies showed that rBSP-(1-75) competitively inhibits the binding of rBSP-(1-100), whereas rBSP-(21-100) inhibits binding to a lesser extent, and rBSP-(43-100) does not inhibit binding. These results suggest that the collagen-binding site of rat BSP is within the sequence 21-42, with residues N-terminal of this region likely also involved. This site was confirmed by the demonstration of collagen-binding activity of a synthetic peptide corresponding to residues 19 -46. The collagen-binding domain, which is highly conserved among species, is enriched in hydrophobic residues and lacks acidic residues. We conclude that residues 19 -46 of BSP represent a novel collagenbinding site. Bone sialoprotein (BSP)1 is a highly anionic phosphoprotein normally expressed only in mineralized tissues and at sites of new mineral formation (1, 2). Studies by nuclear magnetic resonance, circular dichroism (CD), and small angle x-ray scattering have concluded that BSP has a flexible conformation with very little, if any, secondary structure (3-6).BSP has been shown to be involved in cell attachment, cell signaling, hydroxyapatite binding, hydroxyapatite nucleation, and collagen binding (7). It has been proposed that the unstructured, flexible nature of BSP is required for its function as a bridging molecule with multiple binding partners (3). An understanding of the interaction between BSP and collagen is important, as the matrix-mineral relationship in bone is characterized by the presence of hydroxyapatite crystals in the hole zones of the collagen fibrils and by their preferential orientation parallel to the fibril axes (8 -10). To account for this pattern of initial mineral deposition, it is believed that BSP, which has been shown to be a potent nucleator of hydroxyapatite in vitro (5,11,12), is closely associated with type I collagen fibrils.An interaction between BSP and collagen type I has been described previously. BSP, b...