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Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.

[...]as the immune status of a patient or comedications change, a drug that had not appeared immunogenic for many years of treatment could begin to induce an immune response. [...]a marketed drug may exhibit IG for the first time in a new and sensitive target population, such as patients with an autoimmune disease or children. [...]bioinformatic approaches provide a good basis for screening and optimizing compounds, but they cannot be used to manage IG once a protein therapeutic has entered human trials. The most frequent application of PBPK is the prediction of DDIs and the confidence in this approach is such that regulators accept simulations as a substitute for clinical trials and as the basis for label statements. [...]although DDIs still cannot be “engineered out” completely, they can be predicted and managed effectively through virtual trial simulation using models with sufficient mechanistic detail. The QSP model used in the IG Simulator has sufficient mechanistic detail to integrate diverse inputs, including bioinformatics predictions of MHC II binding to antigenic peptides, in vitro cell‐based assays and clinical measurements of compound concentrations, and ADA titers. [...]a detailed simulation of complex immune system interactions allows for

The adaptive immune response starts when CD4+ T cells recognize peptide antigens presented by class II molecules of the Major Histocompatibility Complex (MHCII). Two outstanding features of MHCII molecules are their polymorphism and the ability of each allele to bind a large panoply of peptides. The ability of each MHCII molecule to interact with a limited, though broad, range of amino acid sequences, or “permissive specificity” of binding, is the result of structural flexibility. This flexibility has been identified through biochemical and biophysical studies, and molecular dynamic simulations have modeled the conformational rearrangements that the peptide and the MHCII undergo during interaction. Moreover, there is evidence that the structural flexibility of the peptide/MHCII complex correlates with the activity of the “peptide-editing” molecule DM. In light of the impact that these recent findings have on our ability to predict MHCII epitopes, a review of the structural and thermodynamic determinants of peptide binding to MHCII is proposed.

HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a \"compare-exchange\" sorting algorithm on an available peptide pool. Such a \"third party\"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that DM-mediated peptide release from HLA-DR (DR) is dependent on the presence of exchange peptide. However, DM also promotes a small amount of peptide release in the absence of exchange peptide. Here we show that SDS-PAGE separates purified peptide/DR1 complexes (pDR1) into two conformers whose ratio is peptide K d -dependent. In the absence of exchange peptide, DM only releases peptide from the slower migrating conformer. Addition of exchange peptide converts the DM-resistant conformer to the slower migrating conformer, which is DM labile. Thus, exchange peptide generates a conformer of pDR1 which constitutes the intermediate for peptide exchange and the substrate for DM activity. The resolution of the intermediate favors the highest affinity peptide. However, once folded into the DM-resistant conformer, even low affinity peptides can be presented in the absence of free peptide, broadening the repertoire available for presentation.

There is limited regulatory guidance that outlines the globally acceptable level of individual and total impurities present in peptide and oligonucleotide drug substances that can be supported and accepted during clinical testing. In early clinical development, there is uncertainty regarding the potential toxicological and immunogenicity risk of these impurities relative to the active pharmaceutical ingredient; however, as pharmaceutical development companies move closer to marketing applications, this uncertainty lessens through knowledge gained by clinical and toxicology studies. While these peptide and oligonucleotide related impurities are predicted to be under process control and to have the same safety profile as the parent drug substance, they do not offer any inherent advantages to the patient. Thus, the safety and specification control of these impurities is frequently challenged by regulatory agencies. In support of phase-appropriate control strategies, this manuscript presents a risk-based approach to evaluate the safety of peptide and oligonucleotide impurities from a toxicology and immunogenicity perspective. In many cases, the proposed safety threshold is higher than what is accepted by regulatory bodies, but still is expected to be safe based upon sound toxicological principles which should be the focus for clinical studies. The risk assessment strategies presented here consider the stage of development, indication, potential impact of unintended cross reactivity with endogenous proteins, dose, and frequency of dosing throughout development to inform chemistry manufacturing and control of inherent safety risks associated with API-related impurities. Importantly, for the first time, this manuscript establishes a threshold of immunogenicity concern along with an experimental mitigation plan specifically for peptide impurities as a function of the development phase. Graphical Abstract

The adaptive immune response begins when CD4+ T cells recognize antigenic peptides bound to class II molecules of the Major Histocompatibility Complex (MHCII). The interaction between peptides and MHCII has been historically interpreted as a rigid docking event. However, this model has been challenged by the evidence that conformational flexibility plays an important role in peptide-MHCII complex formation. Thermodynamic analysis of the binding reaction suggests a model of complexation in which the physical-chemical nature of the peptide determines the variability in flexibility of the substates in the peptide-MHC conformational ensemble. This review discusses our understanding of the correlation between thermodynamics of peptide binding and structural features of the resulting complex as well as their impact on HLA-DM activity and on our ability to predict MHCII-restricted epitopes.

We present a systematic, top-down, thermodynamic parametrization scheme for dissipative particle dynamics (DPD) using water-octanol partition coefficients, supplemented by water-octanol phase equilibria and pure liquid phase density data. We demonstrate the feasibility of computing the required partition coefficients in DPD using brute-force simulation, within an adaptive semi-automatic staged optimization scheme. We test the methodology by fitting to experimental partition coefficient data for twenty one small molecules in five classes comprising alcohols and poly-alcohols, amines, ethers and simple aromatics, and alkanes (i.e. hexane). Finally, we illustrate the transferability of a subset of the determined parameters by calculating the critical micelle concentrations of selected alkyl ethoxylate surfactants, in good agreement with reported experimental values.

In this paper we present a novel methodology based on a topological entropy, the so-called persistent entropy, for addressing the comparison between discrete piecewise linear functions. The comparison is certified by the stability theorem for persistent entropy. The theorem is used in the implementation of a new algorithm. The algorithm transforms a discrete piecewise linear function into a filtered simplicial complex that is analyzed with persistent homology and persistent entropy. Persistent entropy is used as discriminant feature for solving the supervised classification problem of real long length noisy signals of DC electrical motors. The quality of classification is stated in terms of the area under receiver operating characteristic curve (AUC=94.52%).

The misalignment between late chronotypes and early school start times affect health, performance and psychological well-being of adolescents. Here we test whether, and how, the baseline chronotype (i.e. chronotype at the beginning of secondary school) and the school timing affect the magnitude and the direction of the developmental change in chronotype during adolescence. We evaluated a sample of Argentinian students (n = 259) who were randomly assigned to attend school in the morning (07:45 a.m.–12:05 p.m.), afternoon (12:40 p.m.–05:00 p.m.) or evening (05:20 p.m.–09:40 p.m.) school timings. Importantly, chronotype and sleep habits were assessed longitudinally in the same group of students along secondary school (at 13–14 y.o. and 17–18 y.o.). Our results show that: (1) although chronotypes partially align with class time, this effect is insufficient to fully account for the differences observed in sleep-related variables between school timings; (2) both school timing and baseline chronotype are independently associated with the direction and the magnitude of change in chronotype, with greater delays related to earlier baseline chronotypes and later school timings. The practical implications of these results are challenging and should be considered in the design of future educational timing policies to improve adolescents’ well-being.