Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from bloodstream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phagedisplayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Softrandomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.The epidermal growth factor receptor (EGF-R) remains one of the best-established targets for anti-tumor therapies. This receptor is involved in cellular processes that contribute to the survival of epithelial cells. Deregulation of the EGF/EGF-R pathway by receptor overexpression or constitutive activation promotes tumor cell proliferation, invasion, and is associated with poor prognosis in cancer 1 . Up to now three anti-EGF-R monoclonal antibodies (mAbs) have been approved for clinical use by the FDA: cetuximab (2004), panitumumab (2006) and necitumumab (2015) 2 . Nimotuzumab recognizes the same target and has a long history of therapeutic use 3 , starting with clinical trials since 1998, and first registered by the Cuban regulatory authorities in 2002 4 . Nimotuzumab is currently approved in Cuba for the treatment of childhood and adult glioma, advanced esophageal cancer, and squamous cell carcinoma of the head and neck, in combination with chemo-radiotherapy or radiotherapy alone, and is also registered in 28 additional countries.The availability of several mAbs targeting the same tumor antigen can result in different clinical outcomes in terms of therapeutic efficacy, safety, anti-tumor mechanisms, immunogenicity, patients' sub-population that can receive a benefit, and development of resistance to therapy. The major molecular determinants behind such complex landscape of clinical effects are the origin of constant domains (species and isotype), the strength of binding to the target (affinity) and the topology of interaction with the specific antigen region that is recognized (epitope specificity). Properties associated to the nature of co...

Activation of T cells triggers the expression of regulatory molecules like the programmed cell death 1 (PD1) protein. The association of PD1 with the natural ligands PDL1 and PDL2 induces an inhibitory signal that prevents T cells from proliferating and exerting effector functions. However, little is known about how the binding of the ligands induce the PD1 inhibitory signal over T cells effector functions. Here, we explore the dynamics of PD1 free, and in complex with different PDL1 variants as well as the therapeutic antibodies nivolumab and pembrolizumab in order to assess the conformational changes in PD1 related to the signaling process. Our simulations suggest a pre‐conformational selection mechanism for the binding of the different PDL1 variants, while an induced‐fit model fits better for the molecular recognition process of the therapeutic antibodies. A deep analysis of the changes on PD1 movement upon the binding to different ligands revealed that as larger is the difference in the conformation adopted by loop C′D with respect to the complex with PDL1 is higher the ligand ability to reduce the PD1 inhibitory signaling. This behavior suggests that targeting specific conformations of this loop can be useful for designing therapies able to recover T cells effector functions.

The activation of T cells is normally accompanied by inhibitory mechanisms within which the PD1 receptor stands out. PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions upon binding the ligands PDL1 and PDL2. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling pathway is activated upon the ligands’ binding remains an open question. In this work, we used molecular dynamics simulations to assess the differences of the PD1 motion in the free state and in complex with the ligands. We found that, in both human and murine systems, the binding of PDL1 and PDL2 stabilizes the conformation of the FG loop similarly. This result, combined with the conservation of the FG loop residues across species, suggests that the conformation of the FG loop is somehow related to the signaling process. We also found a high similarity between the PD1–PDL1 structures with the variable region of an antibody structure, where the FG loop occupies a similar position to the CDR3 light chain.