Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein-Protein Conjugation.

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are hybrid molecules intended to overcome the drawbacks of conventional small molecule chemotherapy and therapeutic antibodies by merging beneficial characteristics of both molecule classes to develop more efficient and patient-friendly options for cancer treatment. During the last decades a versatile bioconjugation toolbox that comprises numerous chemical and enzymatic technologies have been developed to covalently attach a cytotoxic cargo to a tumor-targeting antibody. Microbial transglutaminase (mTG) that catalyzes isopeptide bond formation between proteinaceous or peptidic glutamines and lysines, provides many favorable properties that are beneficial for the manufacturing of these conjugates. However, to efficiently utilize the enzyme for the constructions of ADCs, different drawbacks had to be overcome that originate from the enzyme's insufficiently understood substrate specificity. Within this review, pioneering methodologies, recent achievements and remaining limitations of mTG-assisted assembly of ADCs will be highlighted.

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates.

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein-protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DAinv ). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies.

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering.

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC50 =2-20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50 =2-7 nm), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

Directed Evolution of a Bond-Forming Enzyme: Ultrahigh-Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display.

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.

Swallowing Tablets and Capsules Increases the Risk of Penetration and Aspiration in Patients with Stroke-Induced Dysphagia.

We evaluated the prevalence of difficulties swallowing solid dosage forms in patients with stroke-induced dysphagia and whether swallowing tablets/capsules increases their risk of penetration and aspiration. Concurrently, we explored whether routinely performed assessment tests help identify patients at risk. Using video endoscopy, we evaluated how 52 patients swallowed four different placebos (round, oval, and oblong tablets and a capsule) with texture-modified water (TMW, pudding consistency) and milk and rated their swallowing performance according to the Penetration Aspiration Scale (PAS). Additionally, Daniels Test, Bogenhausener Dysphagiescore, Scandinavian Stroke Scale, Barthel Index, and Tinetti's Mobility Test were conducted. A substantial proportion of the patients experienced severe difficulties swallowing solid oral dosage forms (TMW: 40.4 %, milk: 43.5 %). Compared to the administration of TMW/milk alone, the placebos increased the PAS values in the majority of the patients (TMW: median PAS from 1.5 to 2.0; milk: median PAS from 1.5 to 2.5, each p value <0.0001) and residue values were significantly higher (p < 0.05). Whereas video-endoscopic examination reliably identified patients with difficulties swallowing medication, neither patients' self-evaluation nor one of the routinely performed bedside tests did. Therefore, before video-endoscopic evaluation, many drugs were modified unnecessarily and 20.8 % of these were crushed inadequately, although switching to another dosage form or drug would have been possible. Hence, safety and effectiveness of swallowing tablets and capsules should be evaluated routinely in video-endoscopic examinations, tablets/capsules should rather be provided with TMW than with milk, and the appropriateness of "non per os except medication" orders for dysphagic stroke patients should be questioned.

Two techniques to make swallowing pills easier.

To evaluate whether 2 techniques (the pop-bottle method for tablets and the lean-forward technique for capsules) ease swallowing of tablets and capsules, we conducted a cross-sectional study including 151 adults of the general German population. Participants swallowed 16 differently shaped placebos, rated their ease of swallowing on an 8-point Likert scale, and swallowed the 2 dosage forms that they had rated most difficult again using the appropriate technique. The pop-bottle method substantially improved swallowing of tablets in 59.7% (169/283) and the lean-forward technique for capsules in 88.6% (31/35). Both techniques were remarkably effective in participants with and without reported difficulties swallowing pills and should be recommended regularly.

Optimization of near-infrared spectroscopic process monitoring at low signal-to-noise ratio.

An approach for the optimization of near-infrared (NIR) spectroscopic process monitoring at low signal-to-noise ratio is presented. It compromises the combined adjustment of different measurement variables and data pretreatments considering the prediction error, economic aspects of the application, and process constraints. The integration time, light intensity, and number of averaged spectra were varied; their mutual influence on the prediction error of partial least squares (PLS) models (i.e., root-mean-square error of cross-validation (RMSECV)) was evaluated in the laboratory. At low signal levels, the spectral uncertainty had a strong impact on the prediction error. It leveled off with increasing values of all three parameters and was finally dominated by other sources of uncertainty. The experimental findings could be characterized and explained by a mathematical equation, which was deduced from theoretical principles. The knowledge about the interaction of the measurement variables allowed their combined adjustment resulting in a reduced impact of spectral uncertainty on the prediction error (i.e., root-mean-square error of prediction (RMSEP)) without additional costs or process modifications. Moreover, a convenient procedure to compensate the stray light caused by strongly absorbing windows was developed. The whole approach was successfully applied to a challenging process, namely, the NIR inline monitoring of the liquid content of two model substances in a rotating suspension dryer.

Synthetic Integrin-Targeting Dextran-Fc Hybrids Efficiently Inhibit Tumor Proliferation In Vitro.

Herein, we present the design, synthesis, and biological evaluation of novel integrin-targeting molecular hybrids combining RGD peptides and a potent cytotoxin presented on dextran polysaccharides. Based on an aglycosylated Fc as a centerpiece, endosomal-cleavable cytotoxic agent monomethyl auristatin E (MMAE) and dextran as multimerization site were covalently connected by two bioorthogonal enzyme-mediated reactions site-specifically. Decoration of dextran with cyclic RGD peptides, introduced by copper "click" reaction, resulted in the final constructs with the potential to kill integrin-overexpressing tumor cells. We found that these modifications had little impact on the stability of the Fc scaffold and the RGD-bearing construct showed good binding properties of αvß3-expressing U87MG cells. Furthermore, the construct showed a remarkable antiproliferative activity. These results demonstrate the general capability of our design to provoke receptor-mediated endocytosis upon binding to the cellular surface, followed by endosomal cleavage of the linkage between Fc-dextran and MMAE and its subsequent release. Our approach opens new avenues to transcribe small molecule binders into tailor-made multimeric molecular hybrids with antitumor potential.

Dextramabs: A Novel Format of Antibody-Drug Conjugates Featuring a Multivalent Polysaccharide Scaffold.

Antibody-drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody-drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug-to-antibody ratios, remarkable hydrophilicity, and high toxicity in vitro.

Quantification of DNA binding, uptake, transmission and expression in bovine sperm mediated gene transfer by RT-PCR: effect of transfection reagent and DNA architecture.

In this study, we compared the transfection effectiveness of liposomes with the new transfection reagent FuGene 6 in bovine sperm mediated gene transfer (SMGT). Furthermore, we examined whether plasmid architecture affects overall efficiency by comparing two plasmids, one of them bearing an additional murine nontranscribed spacer (nts) insert (CMV-INF-tau-IRES-EGFP versus CMV-INF-tau-IRES-EGFP-nts). To accomplish that, we quantified plasmid binding and uptake to spermatozoon and transfer and expression of foreign DNA into embryos by real time PCR. More plasmids bound to spermatozoa when treated with FuGene 6 than with liposome treatment (p<0.05) reaching highest counts in plasmids bearing the nts sequence (p<0.05). Mean number of plasmids taken up was significantly (p<0.05) affected by transfection strategy (1-3 versus 15-81 versus 120-162) with plasmids bearing the nts sequence being 2-8 fold more effective (p<0.05). Culture of SMGT derived embryos up to day 9 did not result in any difference in terms of cleavage rate (64.2-84.2%) and development to blastocyst stage (18.8-26.3%) between different groups. Insert of the nts fragment significantly (p<0.05) affected mean number of transmitted plasmids to 4-cell stage embryos (44 versus 7) and relative INF-tau mRNA expression level in day 9 blastocysts (7-8 fold). However, only six blastocysts (3.6%) exhibited green fluorescence indicating low EGFP protein production. In conclusion, we were able to show effectiveness of sperm mediated gene transfer is significantly affected by choice of transfection reagent and by plasmid architecture.

[Legal considerations of telemedical care]. / Rechtliche Rahmenbedingungen der telemedizinischen Versorgung.

Telemedical methods are on the rise in patient care. In addition to the actual changes for both sides in the physician-patient relationship, the use of information and communication technology also involves legal challenges. This article deals with the legal framework of telemedical care. Thereby the article discusses the prohibition of remote treatment (§ 7 Abs. 4 MBO-Ä) and the question to what extent the omission of a telemedical method of treatment can fulfill a medical breach of duty. A distinction must be drawn between the question as to whether telemedical monitoring shall be executed and the question how to use telemedical systems, in order to mitigate liability risks for the physicians. The physician can for example violate his/her medical duties by not sufficiently informing the patient about all essential circumstances concerning the consent, by not adequately monitoring the functioning of the telemedical devices, or by not reacting fast enough to telemedical occurrence reports. All this may lead to a case of liability.

Bovine blastocyst diameter as a morphological tool to predict embryo cell counts, embryo sex, hatching ability and developmental characteristics after transfer to recipients.

The aim of the present study was to explore whether the blastocyst diameter and the zona thickness at 168 h after fertilisation are useful parameters to predict quality and viability of bovine in-vitro-produced (IVP)-embryos. Although significant (P

Effects of different laser-drilled openings in the zona pellucida on hatching of in vitro-produced cattle blastocysts.

OBJECTIVE: To examine whether the size of artificial openings in the zona pellucida effects the hatching process in in vitro-produced cattle embryos. DESIGN: We evaluated the course of hatching of cattle blastocysts with and without laser-drilled zona pellucida openings of different sizes. SETTING: University-based cattle reproductive biotechnology laboratory. INTERVENTION(S): Cattle blastocysts were produced in vitro. Laser treatment was performed 7 days after fertilization. In group I, a small opening of 7-15 microm in width and 40 microm in length was created. In group II, an opening of at least 40 by 40 microm was created. Group III blastocysts received no laser treatment. MAIN OUTCOME MEASURE(S): Hatching course, zona pellucida thickness, diameter. RESULT(S): By day 9, the rate of hatched blastocysts was 27.1%, 63,0%, and 42,9% in groups I, II, and III, respectively. The remainder in group I were entrapped during hatching in a typical figure eight shape. The hatching rate in group II was significantly different from the other groups. All laser-treated blastocysts (groups I and II) showed no change of zona pellucida diameter until day 9. CONCLUSION(S): These results suggest that the size of the artificial openings in the zona pellucida has an influence on the hatching process.

A new approach for freezing of aqueous solutions under active control of the nucleation temperature.

An experimental setup for controlled freezing of aqueous solutions is introduced. The special feature is a mechanism to actively control the nucleation temperature via electrofreezing: an ice nucleus generated at a platinum electrode by the application of an electric high voltage pulse initiates the crystallization of the sample. Using electrofreezing, the nucleation temperature in pure water can be precisely adjusted to a desired value over the whole temperature range between a maximum temperature Tn(max) close to the melting point and the temperature of spontaneous nucleation. However, the presence of additives can inhibit the nucleus formation. The influence of hydroxyethylstarch (HES), glucose, glycerol, additives commonly used in cryobiology, and NaCl on Tn(max) were investigated. While the decrease showed to be moderate for the non-ionic additives, the hindrance of nucleation by ionic NaCl makes the direct application of electrofreezing in solutions with physiological salt concentrations impossible. Therefore, in the multi-sample freezing device presented in this paper, the ice nucleus is produced in a separate volume of pure water inside an electrode cap. This way, the nucleus formation becomes independent of the sample composition. Using electrofreezing rather than conventional seeding methods allows automated freezing of many samples under equal conditions. Experiments performed with model solutions show the reliability and repeatability of this method to start crystallization in the test samples at different specified temperatures. The setup was designed to freeze samples of small volume for basic investigations in the field of cryopreservation and freeze-drying, but the mode of operation might be interesting for many other applications where a controlled nucleation of aqueous solutions is of importance.