Genetically engineered fusion proteins for treatment of cancer.

In this review, we summarize approaches to treat cancer with genetically engineered fusion proteins. Such proteins can act as decoy receptors for several ligands or as recruiters of immune effector cells to tumor. Examples of interference with growth factor-mediated tumor growth and tumor-related angiogenesis with fusion proteins consisting of the extracellular domains, and in some cases also of entities of one or several receptors and the Fc part of human IgG1, are discussed. In addition, we present strategies for recruitment of immune effector cells to tumor with fusion proteins. This can be achieved with fusion proteins consisting of a tumor-related antibody and a cytokine or major histocompatibilty complex class-I-peptide complexes, by T-cell receptor cytokine fusion proteins or by combination of a T-cell-recruiting antibody with a tumor-related ligand or a defined T-cell receptor.

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing.

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

Superior serum half life of albumin tagged TNF ligands.

Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

Measurement of left ventricular velocities: phase contrast MRI velocity mapping versus tissue-doppler-ultrasound in healthy volunteers.

The aim of this study was the comparison of phase contrast magnetic resonance imaging (PCMRI) measurements of left ventricular velocities in a physiological in vivo setting with tissue-Doppler-ultrasound (tissue Doppler imaging: TDI) data in healthy volunteers. Images were acquired in short axis view using a flow compensated black blood k-space segmented gradient echo sequence. Velocity encoding was performed by adding a bipolar gradient after each rf-pulse to the otherwise identical pulse sequences. Full in-plane velocity information of the moving heart was obtained in 16 heartbeats within one breath-hold measurement. Twenty-nine healthy volunteers (mean age=25 years) were examined with both imaging modalities. Both PCMRI and TDI demonstrate a biphasic profile of radial velocities over the cardiac cycle. Intraindividual comparison of left ventricular velocity data acquired using PCMRI and TDI show a very good correspondence with r-values of 0.97. The in vivo study in 29 healthy volunteers demonstrates a high validity of time-resolved phase contrast measurements for the analysis of left ventricular myocardial velocities.

Fast phase contrast cardiac magnetic resonance imaging: improved assessment and analysis of left ventricular wall motion.

PURPOSE: To evaluate the use of CINE phase contrast magnetic resonance imaging (MRI) to assess and characterize left ventricular wall motion by two- or three-directional velocity vector fields that reflect the temporal evolution of myocardial velocities over the whole cardiac cycle. MATERIAL AND METHODS: A fast imaging protocol is presented that permits the assessment of the pixel-wise full in-plane velocity information of the beating heart within a single breath-hold measurement. Temporal resolution of the acquired images is improved by the use of high-speed gradients and application of view sharing to black blood k-space segmented gradient echo imaging. A novel tool for data analysis is presented based on correlating locally different myocardial motion patterns to averaged left ventricular velocities reflecting nonpathological myocardial function. RESULTS: Measurement protocol and postprocessing options were evaluated in a study with 16 normal volunteers. Simulations showed that correlation analysis can be used to differentiate regions with altered velocity waveforms from global radial velocities. Results of patient examinations are presented on an exemplary basis and demonstrate that correlation analysis provides an effective method for identification and classification of myocardial dynamics. CONCLUSION: Within the framework of our volunteer and patient examinations, fast phase contrast cardiac MRI has proven to be a reliable method to assess and analyze myocardial performance on the basis of two-directional velocity vector fields.