Molecular modelling of the mass density of single proteins

<div><p>Using molecular dynamics (MD) simulations, the density of single proteins and its temperature dependence was modelled starting from the experimentally determined protein structure and a generic, transferable force field, without the need of prior parameterization. Although all proteins consist of the same 20 amino acids, their density in aqueous solution varies up to 10% and the thermal expansion coefficient up to twofold. To model the protein density, systematic MD simulations were carried out for 10 proteins with a broad range of densities (1.32–1.43 g/cm³) and molecular weights (7–97 kDa). The simulated densities deviated by less than 1.4% from their experimental values that were available for four proteins. Further analyses of protein density showed that it can be essentially described as a consequence of amino acid composition. For five proteins, the density was simulated at different temperatures. The simulated thermal expansion coefficients ranged between 4.3 and 7.1 × 10⁻⁴ K⁻¹ and were similar to the experimentally determined values of ribonuclease-A and lysozyme (deviations of 2.4 and 14.6%, respectively). Further analyses indicated that the thermal expansion coefficient is linked to the temperature dependence of atomic fluctuations: proteins with a high thermal expansion coefficient show a low increase in flexibility at increasing temperature. A low increase in atomic fluctuations with temperature has been previously described as a possible mechanism of thermostability. Thus, a high thermal expansion coefficient might contribute to protein thermostability.</p> </div

A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties.

Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity

Antitumor Activity of a Mesenchymal Stem Cell Line Stably Secreting a Tumor-Targeted TNF-Related Apoptosis-Inducing Ligand Fusion Protein

Mesenchymal stem cells (MSCs) are currently exploited as gene delivery systems for transient in situ expression of cancer therapeutics. As an alternative to the prevailing viral expression, we here describe a murine MSC line stably expressing a therapeutic protein for up to 42 passages, yet fully maintaining MSC features. Because of superior antitumoral activity of hexavalent TNF-related apoptosis-inducing ligand (TRAIL) formats and the advantage of a tumor-targeted action, we choose expression of a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) as a model. The bioactivity of Db-scTRAIL produced from an isolated clone (MSC.TRAIL) was revealed from cell death induction in Colo205 cells treated with either culture supernatants from or cocultured with MSC.TRAIL. In vivo, therapeutic activity of MSC.TRAIL was shown upon peritumoral injection in a Colo205 xenograft tumor model. Best antitumor activity in vitro and in vivo was observed upon combined treatment of MSC.TRAIL with bortezomib. Importantly, in vivo combination treatment did not cause apparent hepatotoxicity, weight loss, or behavioral changes. The development of well characterized stocks of stable drug-producing human MSC lines has the potential to establish standardized protocols of cell-based therapy broadly applicable in cancer treatment

Determination binding kinetics by quartz crystal microbalance measurements.

<p>Specificity and affinity of scDb-SpG_C3, scDb-SpG_C3Fab, and scDb-SpG_C3FabRR for human and mouse serum IgG, Fc Fragments, and Fab fragments were determined by QCM measurements at pH 7.4. Two-fold dilutions were added, starting at a concentration of 1,000 nM.</p

Fab and Fc binding sites in SpG_C3.

<p>a) Alignment of the different SpG IgBDs (C1-C3) as well as SpG_C3Fab with mutated Fc binding site. The position of β-strands and the α-helix is indicated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139838#pone.0139838.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139838#pone.0139838.ref019" target="_blank">19</a>]. Positions randomized in the phage library are indicated by orange triangles (residues 33, 35, 36, and 37). b) Structure of SpG_C3 (pdb entry 1IGC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139838#pone.0139838.ref017" target="_blank">17</a>]). The Fc binding site is shown in red/yellow, the major Fab binding site in blue, and the minor Fab binding site in yellow, overlapping with the Fc binding site. Residues mutated to alanines are indicated as red spheres, positions mutated in the phage library are highlighted as orange spheres. c) Superposition of SpGC3 bound to an Fc fragment (pdb entry 1FCC) and a Fab fragment (1IGC) (variable domains are not shown). Residues are marked as in b).</p

Affinity determination by QCM (K_D values in nM).

<p>Affinity determination by QCM (K_D values in nM).</p

Effector cell retargeting and activation by bispecific scDb fusion proteins determined by IL–2 release.

<p>CEA positive target cells (LS174T) were incubated with scDb-SpG_C3 variants together with or without 100 μg/ml human IgG 1 h prior to addition of PBMCs. Cell free supernatant was analyzed after 24 h for released IL-2 by ELISA.</p

Biochemical characterization of antibody-IgBD fusion proteins.

<p>a) Composition of the bispecific scDb-IgBD and the monovalent scFv-IgBD fusion protein. b) SDS-PAGE analysis and HPLC size exclusion chromatography of scDb (1), scDb-SpG_C3 (2), scDb-SpG_C3Fab (3), scDb-SpG_C3FabRR analyzed under reducing conditions. c) SDS-PAGE analysis and HPLC size exclusion chromatography of scFv (1), scFv-SpG_C3 (2), scFv-SpG_C3Fab (3), scFv-SpG_C3FabRR analyzed under reducing conditions.</p

Pharmacokinetics of the fusion proteins in CD1 mice.

<p>a) scDb fusion proteins. b) scFv fusion proteins. 25 μg of scDb-SpG_C3 (a) or scFv-SpG_C3 (b) variants was injected intravenously into female CD1 mice. Serum concentrations were determined by ELISA against CEA and data were normalized considering the maximal concentration at the first time point (3 min).</p

Pharmacokinetic properties (n = 3–6).

<p>Pharmacokinetic properties (n = 3–6).</p