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Antibody-based therapeutics for treatment of various tumors have grown rapidly in recent years. Unfortunately, safety issues, attributed to off-tumor effects and cytotoxicity, are still a significant concern with the standard of care. Improvements to ensure targeted delivery of antitumor pharmaceuticals are desperately needed. We previously demonstrated that incorporating histidyl pH-switches in an anti-HER2 antibody induced selective antigen binding under acidic pH conditions (MAbs 2020;12:1682866). This led to an improved safety profile due to preferential targeting of the oncoprotein in the acidic solid tumor microenvironment. Following this success, we expanded this approach to a set of over 400 antibody structures complexed with over 100 different human oncoproteins, associated with solid tumors. Calculations suggested that mutations to His of certain residue types, namely Trp, Arg, and Tyr, could be significantly more successful for inducing pH-dependent binding under acidic conditions. Furthermore, 10 positions within the complementarity-determining region were also predicted to exhibit greater successes. Combined, these two accessible metrics could serve as the basis for a sequence-based engineering of pH-selective binding. This approach could be applied to most anticancer antibodies, which lack detailed structural characterization.

The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform guides the selection of mutants that improve/modulate the affinity of antibodies and other biologics. Predicted affinities are based on a consensus z-score from three scoring functions. Computational predictions are interleaved with experimental validation, significantly enhancing the robustness of the design and selection of mutants. A key step is an initial exhaustive virtual single-mutant scan that identifies hot spots and the mutations predicted to improve affinity. A small number of proposed single mutants are then produced and assayed. Only the validated single mutants (i.e., having improved affinity) are used to design double and higher-order mutants in subsequent rounds of design, avoiding the combinatorial explosion that arises from random mutagenesis. Typically, with a total of about 30-50 designed single, double, and triple mutants, affinity improvements of 10- to 100-fold are obtained.

The design of superior biologic therapeutics, including antibodies and engineered proteins, involves optimizing their specific ability to bind to disease-related molecular targets. Previously, we developed and applied the Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform for virtual affinity maturation of antibodies (Vivcharuk et al. in PLoS One 12(7):e0181490,https://doi.org/10.1371/journal.pone.0181490, 2017). However, ADAPT is limited to point mutations of hot-spot residues in existing CDR loops. In this study, we explore the possibility of wholesale replacement of the entire H3 loop with no restriction to maintain the parental loop length. This complements other currently published studies that sample replacements for the CDR loops L1, L2, L3, H1 and H2. Given the immense sequence space theoretically available to H3, we focused on the virtual grafting of over 5000 human germline-derived H3 sequences from the IGMT/LIGM database increasing the diversity of the sequence space when compared to using crystalized H3 loop sequences. H3 loop conformations are generated and scored to identify optimized H3 sequences. Experimental testing of high-ranking H3 sequences grafted into the framework of the bH1 antibody against human VEGF-A led to the discovery of multiple hits, some of which had similar or better affinities relative to the parental antibody. In over 75% of the tested designs, the re-designed H3 loop contributed favorably to overall binding affinity. The hits also demonstrated good developability attributes such as high thermal stability and no aggregation. Crystal structures of select re-designed H3 variants were solved and indicated that although some deviations from predicted structures were seen in the more solvent accessible regions of the H3 loop, they did not significantly affect predicted affinity scores.

This article seeks to provide a comparative and up-to-date overview of the applicable rules and relevant practice of the European Court of Human Rights and of the United Nations Human Rights Committee on forum duplication in international human rights litigation. While specific inadmissibility clauses have been included in both the European Convention on Human Rights and the Optional Protocol to the International Covenant on Civil and Political Rights with a view to preventing multiple human rights petitions in relation to the same matter, their respective scopes differ. Moreover, the applicable normative framework has led to important—and diverging—judicial developments in Strasbourg and in Geneva, which may be of great significance in human rights practice and therefore deserve to be thoroughly addressed.

Requirements Engineering (RE) covers not only the capture and structuring of various properties the system should achieve but also the identification of high-level choices on how to achieve such goals or to avoid related obstacles. Generic RE frameworks support simple formalisation of alternatives using AND/OR refinements while more specialised fields such as safety and security engineering have richer analysis capabilities respectively through fault and attack trees. In this paper, we review the various constructs proposed in those domains and state their semantics at RE level to support safety and security co-engineering. As a supplementary step, we propose a mapping on the semantics provided by Constraint Programming in order to search for optimal configurations in the design space of a RE model. We consider multiple objectives stated as non-functional requirements and formalised using quantified attributes over goal models. Our work is validated on the complex design of an oil pipe system mixing safety and security critical properties.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

Small and medium-sized enterprises SMEs involved in software development often experience problems in mastering their development processes. This can lead to time and cost overruns or failure to address functional and nonfunctional requirements. In general, this can significantly affect customer satisfaction and hamper business growth potential. This paper reports on a survey conducted in Belgium to assess more precisely which and how SMEs are affected by problems related to development processes. The survey was driven by the ISO29110 lightweight standard focusing on very small entities developing software, ie, whose internal IT department is less than 25 people. Worldwide, and in particular in Belgium, this represents a very large portion of SMEs in business. Survey results highlight the most frequent issues and how they may be linked to organization and project characteristics. The survey is based on a free online self-assessment tool. Therefore, results go 1 step further than identifying issues encountered in companies: the tool also infers a set of quick-win high-level recommendations to solve these issues. Our results are also compared with those reported by other surveys targeting both large and small companies.

Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

Placer is a model-based tool that, given a model of heterogeneous (or at least multi-core) hardware and a task-based complex software, finds a mapping of the software tasks on the various processing elements of the hardware, together with a routing of the transmissions on the available busses, and provides a schedule for the tasks and transmissions. The mapping can minimize either the run time, energy consumption, or both in a multi-objective fashion. The tool combines an Eclipse front-end and a web-service placement back-end based on the OscaR constraint programming library. The tool demonstration illustrates how to build the task model, perform the analysis and possibly iterate to improve the placement.

Despite decades of development, protein–protein docking remains a largely unsolved problem. The main difficulties are the immense space spanned by the translational and rotational degrees of freedom and the prediction of the conformational changes of proteins upon binding. FFT is generally the preferred method to exhaustively explore the translation-rotation space at a fine grid resolution, albeit with the trade-off of approximating force fields with correlation functions. This work presents a direct search alternative that samples the states in Cartesian space at the same resolution and computational cost as standard FFT methods. Operating in real space allows the use of standard force field functional forms used in typical non-FFT methods as well as the implementation of strategies for focused exploration of conformational flexibility. Currently, a few misplaced side chains can cause docking programs to fail. This work specifically addresses the problem of side chain rearrangements upon complex formation. Based on the observation that most side chains retain their unbound conformation upon binding, each rigidly docked pose is initially scored ignoring up to a limited number of side chain overlaps which are resolved in subsequent repacking and minimization steps. On test systems where side chains are altered and backbones held in their bound state, this implementation provides significantly better native pose recovery and higher quality (lower RMSD) predictions when compared with five of the most popular docking programs. The method is implemented in the software program ProPOSE (Protein Pose Optimization by Systematic Enumeration).

Small and medium-sized enterprises (SMEs) play a key role in the worldwide economy and are increasingly dependent on software as part of their products/services or to support their operation (e.g. e-commerce, smart manufacturing). Over the past 15 years, as a research and technology transfer centre, CETIC has been busy helping Belgian SMEs to increase their maturity in software development. We have also contributed specific methods and tools for the SME target, including the OWPL framework and now the ISO29110 standard. This talk aims at sharing what we learned from different types of SMEs (from startups to grownups, both IT and non IT) about common problems gathered in a long term survey. We then focus on key issues like requirements, technical debt, test/release and (agile/lean) project management. Finally, we share our thoughts on new challenges ahead raised by the ever increasing connectivity like cybersecurity and privacy regulation (GDPR).

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.

As our world is going digital, Electronic Evidence are increasingly used in court and not just for computer related crimes. At the same time, organised crimes are also following the globalisation trend and often require fast cooperation across judicial authorities of several countries to be efficiently fought. However this is currently difficult to achieve because the integrity of electronic evidence can easily be compromised and legislation is often lagging behind in their use of ICT means. The context of this work is to develop a roadmap for the collection, use and exchange of electronic evidence across EU Member states. In this paper we report about a detailed requirements analysis re-lying on the GRL goal-oriented method. We present strategic rationales underlying the management of electronic evidence and refine them into specific requirements that can be achieved by existing or enhanced systems like I-Link and TESTA as well as data exchange frameworks such as CybOX, DFAX and UMF.

Cloud application deployment is becoming increasingly popular for the removal of upfront hardware costs, the pay-per-use cost model and their ability to scale. However, deploying software on the Cloud carries both opportunities and threats regarding energy efficiency. In order to help Cloud application developers learn and reason about the energy consumption of their application on the server-side, we have developed a framework centred on a UML profile for relating energy goals, requirements and associated KPI metrics to application design and deployment elements. Our previous work has focused on the use of such a framework to carry out our run-time experiments in order to select the best approach. In this paper, we explore the feasibility of a complementary approach for providing support at design time based on finer grained deployment models, the specification of Cloud and energy adaptation policies and the use of a discrete event simulator for reasoning on key performance indicators such as energy but also overall performance, delay and costs. The goal is to support the Cloud developer in pre-selecting the best trade-off that can be further tuned at run-time.

ICT energy efficiency is a growing concern. A great effort was already done making hardware more energy efficient and aware. Although a part of that effort is devoted to specific software areas like embedded/mobile systems, much remains to be done at the software level, especially for applications deployed in the Cloud. There is a increasing need to help Cloud application developers to learn to reason about how much energy is consumed by their applications on the server-side. This paper presents a set of tools which guides the developers of Cloud applications in key steps. First, at requirements stage, in order to capture energy goals in a measurable way and relate them with important Non-Functional Requirements (NFR). Second, at design level, an UML profile supporting energy Key Performance Indicators (KPI) is used in order to keep tracking off those goals and metrics across the functional design of the application. Third, at runtime, measurements probes are automatically deployed and the collected data is processed in order to be analysed at the previously goal level. Specific tools for analysing the energy behaviour and helping in making a choice among different design alternatives are also proposed.

Today, it is not seriously challenged that human rights law applies to proceedings before the International Criminal Court. The exact boundaries of this statement, however, might be less clear. The present article argues that the extent of applicability of human rights law cannot be precisely described unless the specific nature of the Court and of international criminal justice in general is taken into consideration. More concretely, it will be demonstrated that the exact scope of applicability of human rights standards to the ICC setting can only be addressed by referring to inherent characteristics (both of the Court and of the international criminal system as a whole) that could possibly bear a reductive impact on that scope. It will be argued throughout the analysis that several of these specific features are indeed capable of reducing the level of protection, while on a closer look others do not display such influence.

The Tol-Pal system of Escherichia coli is a macromolecular complex located in the cell envelope. It is involved in maintaining the integrity of the outer membrane and is required for the uptake of two different types of macromolecules, which are bacteriotoxins (colicins) and DNA of filamentous bacteriophages. The TolA protein plays a central role in these import mechanisms. Its C-terminal domain (TolAIII) is involved in the translocation step via direct interaction with the N-terminal domain of colicins and the N-terminal domain of the phage minor coat gene 3 protein (g3pN1). Extreme behaviours of TolAIII have been previously observed, since the structure of TolAIII either remained unaffected or adopted disordered conformation upon binding to different pore-forming colicins. Here, we have solved the 3D structure of free TolAIII by heteronuclear NMR spectroscopy and compared it to the crystal structure of TolAIII bound to g3pN1 in order to study the effect of g3pN1 on the tertiary structure of TolAIII. Backbone 1H, 15N and 13C resonances of the g3pN1-bound TolAIII were also assigned and used to superimpose the solution structure of free TolAIII on the crystal structure of the g3pN1-TolAIII fusion protein. This allowed us to track conformational changes of TolAIII upon binding. While the global fold of free TolAIII is mainly identical to that of g3pN1-bound TolAIII, shift of secondary structures does occur. Thus, TolAIII, which interacts also in vivo with Pal and TolB, is able to adapt its conformation upon binding to various partners. Possible models for protein binding mechanisms are discussed to explain this so-far unobserved behaviour of TolAIII.