Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions, while retaining their specific mode of action. Gaining a deeper understanding of the drivers of instability across different stress conditions, will potentially enable the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic molecular dynamics simulations under two different stresses of low pH and high temperature. RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding was initiated through loss of contacts at the constant domain interface (CL-CH1), prior to CL domain unfolding. By contrast, thermal unfolding began with loss of contacts in both the CL-CH1 and variable domain interface (VL-VH), followed by domain unfolding of CL and also of VH, thus revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the CL-CH1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, packing density calculations found these residues to be under-packed relative to other inter-domain residues. Two salt bridges were identified that possibly drive the conformational change at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis of existing Fab sequences revealed considerable scope for further engineering, where certain natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR), which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for future protein engineering work aimed at stabilizing to both thermal and pH-stresses simultaneously

Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5 Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5–7% loss of native contacts in all four domains, and simultaneous loss of >30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Computationally-guided semi-rational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilise proteins to aggregation under some conditions, previous studies suggest that such an approach is limited because thermal transition temperatures (Tm) and the fraction of protein unfolded (fT) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the Tm. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, that reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilising mutations to regions of the A33 Fab surface structure, that were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations, and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation, and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔGND for each potential point mutation. Additional destabilising variants were prepared as controls to evaluate the prediction accuracy, and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of eighteen variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased Tm values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by Tm. However, six of the twelve stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate

L'atteinte du sinus caverneux dans le carcinome adenoïde systique

Introduction: Head and neck adenoid cystic carcinoma (ACC) is a malignant epithelial neoplasm, developing from salivary gland tissue. Cavernous sinus and skull base invasion by perinervous spread is a particular behavior of this tumor entity. We report two cases of cavernous sinus invasion.Cases report: The first one was originated in the parotid gland, invaded infratemporal fossa and extended to cavernous sinus through perineural infiltration along trigeminal nerve. Second case of ACC was developed in submandibular gland. An invasion of cavernous sinus was observed two years later despite a surgical resection with post operative  chemoradiotherapy.Discussion: ACC is characterized by locally aggressive invasion and a strong tendency to recur. Perineural spread extension is common in ACC, as a precursor of skull base invasion and cavernous sinus involvment along the mandibular, maxillary and facial nerves. Magnetic resonance imaging (MRI) has a higher sensitivity and specificity in detecting perineural spread. Surgical therapy is the choice treatment although complete resection is not possible in all cases due to the infiltration andperineural spread. So, radiation therapy is considered effective, and has been used successfully in ACC invading cavernous sinus. The place of chemotherapy has yet to be determinate. The prognosis of these extensive tumors is bad because high frequency of local recurrence and distant metastases.Key-words: adenoid cystic carcinoma, cavernous sinus, skull base, perineural invasion

Many-core Branch-and-Bound for GPU accelerators and MIC coprocessors

International audienceCoprocessors are increasingly becoming key building blocks of High Performance Computing platforms. These many-core energy-efficient devices boost the performance of traditional processors. On the other hand, Branch-and-Bound (B&B) algorithms are tree-based exact methods for solving to optimality combinatorial optimization problems (COPs). Solving large COPs results in the generation of a very large pool of subproblems and the evaluation of their associated lower bounds. Generating and evaluating those subproblems on coprocessors raises several issues including processor-coprocessor data transfer optimization, vectorization, thread divergence, and so on. In this paper, we investigate the offload-based parallel design and implementation of B&B algorithms for coprocessors addressing these issues. Two major many-core architectures are considered and compared: Nvidia GPU and Intel MIC. The proposed approaches have been experimented using the Flow-Shop scheduling problem and two hardware configurations equivalent in terms of energy consumption: Nvidia Tesla K40 and Intel Xeon Phi 5110P. The reported results show that the GPU-accelerated approach outperforms the MIC offload-based one even in its vectorized version. Moreover, vectorization improves the efficiency of the MIC offload-based approach with a factor of two

Traitement des diplegies laryngees en fermeture: Apport du laser

Introduction: Les diplegies laryngees en fermeture sont rares et mal tolerees par les patients. Leur prise en charge therapeutique a connu de nombreuses avancees au cours des dernieres annees. Le but de ce travail est dfetudier la place du laser dans leur prise en charge.Malades et methodes : Notre etude est retrospective a propos de six patients porteurs de diplegie laryngee en fermeture. Lfage moyen etait de 46 ans (16 et 70 ans). Lfetiologie etait une intubation prolongee ou traumatique dans 3 cas, post thyroidectomie totale dans deux autres cas et idiopathique dans 1 cas. Le traitement a consiste en une tracheotomie en urgence dans 5 cas. Tous les patients ont beneficie dans un secondtemps dfun traitement au laser apres 9 mois de surveillance.Resultats : La cordectomie posterieure a ete realisee en premier temps dans 4 cas. Elle etait bilaterale en deux temps dans 2 cas et unilaterale dans 2 cas. Une aryteno.dectomie initiale a ete realisee pour les deux autres patients. Une revision chirurgicale a consiste en une arytenoidectomie dans 2 cas et une cordotomie partielle posterieure controlaterale dans un cas. La decanulation etait bien toleree pour 4 patients. Un seul malade a garde une gene respiratoire sans veritabledyspnee avec une canule parlante fermee et la decanulation a ete refusee. Une voix satisfaisante a ete obtenue pour tous les malades. Des fausses route transitoires ont ete note dans 2 cas.Discussion : Actuellement, le traitement endoscopique au laser constitue le traitement de choix dans la diplegie laryngee et doit etre propose en premiere intension. Deux techniques principales sont decrites :  lfaryteno.dectomie au laser type Ossof et la cordectomie posterieure transverse de kashima. plusieurs series ont defendu chacune des deux techniques. Notre serie a montre que les deux techniques peuvent etre associees pour donner de meilleurs resultats.Mots cles : diplegie laryngee, Laser, aryteno.dectomie, cordectomie posterieure

Facteurs predictifs de survenue de pharyngostome après laryngectomie totale

Objectif : Déterminer l’incidence de survenue de la fistule pharyngo-cutanée ou pharyngostome après laryngectomie totale ainsi que les facteurs prédisposants.Matériel et méthodes: C’est une étude rétrospective qui a porté sur 187 malades ayant eu une laryngectomie totale à notre département (Janvier 1985-Décembre 2007). Nous avons étudié les facteurs cliniques, histologiques, les modalités thérapeutiques ainsi que évolutives à la recherche de corrélation avec la survenue de pharyngostome après laryngectomie totale. -Résultats : Les pharyngostomes ont été observé dans14,4% des cas. La chimioradiothérapie préopératoire était significativement corrélée à l’apparition de pharyngostome (p=0,008). L’infection postopératoire est un facteur hautement significatif de survenue de pharyngostome (p=0,000). De même que l’hémorragie postopératoire (p=0,016), les troubles de ladéglutition (p=0,037), et les bronchopneumopathies (p=0,032). La rupture capsulaire lors d’un envahissement ganglionnaire était très  significativement corrélée à la survenue d’un pharyngostome (p = 0,001).Conclusion : en présence de facteurs de risque significatifs de  pharyngostome, cette complication peut ainsi être prévue même si sa prévention demeure encore difficile.Mots-clés : Pharyngostome, Laryngectomie totale, Incidence, facteurs prédisposants

Quel bilan d’extension faut-il faire pour les carcinomes indifferencies du nasopharynx ?

Introduction: Nasopharyngeal carcinoma prognosis is often correlated with its local extension but especially lymphatic node and metastatic.The aimof our work was to study sensitivity and the specificity of clinical and paraclinic explorations in the initial assessmentPatients and methods : It .s about a retrospective study of 366 patients having nasopharyngeal carcinoma, diagnosed over eleven years period between 1993 and 2003 in Sfax hospital. Into pretherapeutic, all the patients had a complementary assessment including:- Nasopharyngeal tomodensitometry (TDM), in all the cases, extended to the cervical area in 112 cases and a magnetic resonnance Imagery (MRI) of the nasopharynx and cerebral in 18 cases.- Metastatic assessment: comprising systematically a chest radiography, an abdominal ultrasonography and an osseous scintiscanning. The statistical study comprised a descriptive study and an analytical study.Results : The metastasis diagnosis was retained in 39 cases (10,7%): osseous in 82%, hepatic in 23% and pulmonary in 12,8% of the cases. The tumour was associated to lymph node N3 in 25 cases (64%). At univariate study, we retained the presence of significant difference between the groups of the metastatic and lack metastatic patients for : the male sex, reason for consultation (cervical node, rhinologic signs and otologic signs) and cervical node at the examination.The multivariate analysis for all the factors was without interest. We choose the parameters according to the result of the univariate study, the literature and parameters' found among all patients with discovered CNP. It comes out from this study that the following elements are providers of metastases: age between 40 and 45years, male sex and cervical node N3a stage.Discussion : The assessment of extension is not standardized for all the authors. Indeed, for the study of the pulmonary extension (AJCC)/ (UICC) recommends the systematic practice of the chest radiography. For (NCCN), the practice of chest radiography is only for patients classified at the stage 2 and 3 in WHO classification. For KUMAR, LEUNG and our results, it is recommended systematically to practice the chest radiography . This radiography would be supplemented by a thoracic tomodensitometry with the least suspect lesion. For the hepatic assessment, some recommend the systematic practice of abdominal echography for the advanced nodestages (N3). For others, it will be indicated only for the symptomatic patients. For (AJCC)/ (UICC) abdominal echography is systematic.For the osseous assessment, KRAIPHIBUL recommends the practice of the osseous scintiscanning only for patients having signs of osseous call but LEUNG and SHAM recommend the practice of the osseous scintiscanning only for the patients having cervical node N3.Key words: Nasopharyngeal carcinoma/ extension assessment/ metastasis

An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism

Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states, is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states - notably amyloids - have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle X-ray scattering (SAXS), atomistic molecular dynamics (MD) simulations, single-molecule FRET (smFRET), and aggregation-prone region (APR) predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, Rg, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full molecular dynamics simulations from the same conditions, and located the conformational changes in the native state to the constant domain of the light chain (CL). This CL displacement was independently confirmed using smFRET measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted aggregation-prone region (APR), suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation