The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Characterizing the Pharmacokinetics and Biodistribution of Therapeutic Proteins: An Industry White Paper.

Characterization of the pharmacokinetics and biodistribution of therapeutic proteins (TPs) is a hot topic within the pharmaceutical industry, particularly with an ever-increasing catalog of novel modality TPs. Here, we review the current practices, and provide a summary of extensive cross-company discussions as well as a survey completed by International Consortium for Innovation and Quality members on this theme. A wide variety of in vitro, in vivo and in silico techniques are currently used to assess pharmacokinetics and biodistribution of TPs, and we discuss the relevance of these from an industry perspective, focusing on pharmacokinetic/pharmacodynamic understanding at the preclinical stage of development, and translation to human. We consider that the 'traditional in vivo biodistribution study' is becoming insufficient as a standalone tool, and thorough characterization of the interaction of the TP with its target(s), target biology, and off-target interactions at a microscopic scale are key to understand the overall biodistribution on a full-body scale. Our summary of the current challenges and our recommendations to address these issues could provide insight into the implementation of best practices in this area of drug development, and continued cross-company collaboration will be of tremendous value. SIGNIFICANCE STATEMENT: The Innovation and Quality Consortium Translational and ADME Sciences Leadership Group working group for the absorption, distribution, metabolism, and excretion of therapeutic proteins evaluates the current practices and challenges in characterizing the pharmacokinetics and biodistribution of therapeutic proteins during drug development, and proposes recommendations to address these issues. Incorporating the in vitro, in vivo and in silico approaches discussed herein may provide a pragmatic framework to increase early understanding of pharmacokinetic/pharmacodynamic relationships, and aid translational modeling for first-in-human dose predictions.

Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276.

BACKGROUND: The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. METHODS: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. RESULTS: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. CONCLUSION: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

Prediction of Human Nonlinear Pharmacokinetics of a New Bcl-2 Inhibitor Using PBPK Modeling and Interspecies Extrapolation Strategy.

S 55746 ((S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide) is a new selective Bcl-2 (B-cell lymphoma 2) inhibitor developed by Servier Laboratories and used to restore apoptosis functions in cancer patients. The aim of this work was to develop a translational approach using physiologically based (PB) pharmacokinetic (PK) modeling for interspecies extrapolation to anticipate the nonlinear PK behavior of this new compound in patients. A PBPK mouse model was first built using a hybrid approach, defining scaling factors (determined from in vitro data) to correct in vitro clearance parameters and predicted Kp (partition coefficient) values. The qualification of the hybrid model using these empirically determined scaling factors was satisfactorily completed with rat and dog data, allowing extrapolation of the PBPK model to humans. Human PBPK simulations were then compared with clinical trial data from a phase 1 trial in which the drug was given orally and daily to cancer patients. Human PBPK predictions were within the 95% prediction interval for the eight dose levels, taking into account both the nonlinear dose and time dependencies occurring in S 55746 kinetics. Thus, the proposed PK interspecies extrapolation strategy, based on preclinical and in vitro information and physiologic assumptions, could be a useful tool for predicting human plasma concentrations at the early stage of drug development.

Molecular dynamics simulations of site point mutations in the TPR domain of cyclophilin 40 identify conformational states with distinct dynamic and enzymatic properties.

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 µs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.

Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells.

Insights into the conformations of three structurally diverse proteins: cytochrome c, p53, and MDM2, provided by variable-temperature ion mobility mass spectrometry.

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.

Nanosensing protein allostery using a bivalent mouse double minute two (MDM2) assay.

The tumor suppressor protein, p53, is either mutated or absent in >50% of cancers and is negatively regulated by the mouse double minute (MDM2) protein. Understanding and inhibition of the MDM2-p53 interaction are, therefore, critical for developing novel chemotherapeutics, which are currently limited because of a lack of appropriate study tools. We present a nanosensing approach to investigate full-length MDM2 interactions with p53, thus providing an allosteric assay for identifying binding ligands. Surface-enhanced Raman scattering (SERS)-active nanoparticles, functionalized with a p53 peptide mimic (peptide 12.1), display biologically specific aggregation following addition of MDM2. Nanoparticle assembly is competitively inhibited by the N-terminal MDM2-binding ligands peptide 12.1 and Nutlin-3. This study reports nanoparticle assembly through specific protein-peptide interactions that can be followed by SERS. We demonstrate solution-based MDM2 allosteric interaction studies that use the full-length protein.

A physiologically based modeling strategy during preclinical CNS drug development.

Physiologically based pharmacokinetic (PBPK) modeling of the central nervous system (CNS) provides the opportunity to predict the relevant drug concentrations at the therapeutic target site during preclinical and clinical development. In order to successfully interpret model results, and to provide confidence in the subsequent human predictions, it is essential that an appropriate model structure is chosen at the preclinical stage which takes into account both physiological and drug-specific knowledge. However, the models published to date in the literature show significant variation in the approaches applied by different authors, which can lead to difficulties in the interpretation of model parameter estimates. We aimed to develop a coherent PBPK modeling approach in the rat, which would also be adaptable depending on the quantity and quality of in vivo data obtained during drug development. Based on a sensitivity analysis of the model parameters, and using three CNS drugs as case studies (atomoxetine, acetaminophen, and S 18986), we proposed a decision tree to aid in the appropriate parametrization and structure of the model according to the data available. We compared our parameter estimates to those originally published, and considered the impact of the respective approaches on the mechanistic interpretation of the parameter values. Since the measurement of brain extracellular fluid (ECF) concentrations using microdialysis is not routinely performed in the industrial environment, we also evaluated the bottom-up scaling of in vitro permeability data from the Caco-2 cell line to predict BBB passive permeability in the absence of measured ECF concentrations. Our strategy demonstrates the value of PBPK as a prediction tool throughout the development process of CNS-targeting drugs.

DNA-binding regulates site-specific ubiquitination of IRF-1.

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

Comparing translational population-PBPK modelling of brain microdialysis with bottom-up prediction of brain-to-plasma distribution in rat and human.

The prediction of brain extracellular fluid (ECF) concentrations in human is a potentially valuable asset during drug development as it can provide the pharmacokinetic input for pharmacokinetic-pharmacodynamic models. This study aimed to compare two translational modelling approaches that can be applied at the preclinical stage of development in order to simulate human brain ECF concentrations. A population-PBPK model of the central nervous system was developed based on brain microdialysis data, and the model parameters were translated to their corresponding human values to simulate ECF and brain tissue concentration profiles. In parallel, the PBPK modelling software Simcyp was used to simulate human brain tissue concentrations, via the bottom-up prediction of brain tissue distribution using two different sets of mechanistic tissue composition-based equations. The population-PBPK and bottom-up approaches gave similar predictions of total brain concentrations in both rat and human, while only the population-PBPK model was capable of accurately simulating the rat ECF concentrations. The choice of PBPK model must therefore depend on the purpose of the modelling exercise, the in vitro and in vivo data available and knowledge of the mechanisms governing the membrane permeability and distribution of the drug.

Molecules targeting a novel homotrimer cavity of Spike protein attenuate replication of SARS-CoV-2.

The SARS-CoV-2 Spike glycoprotein (S) utilizes a unique trimeric conformation to interact with the ACE2 receptor on host cells, making it a prime target for inhibitors that block viral entry. We have previously identified a novel proteinaceous cavity within the Spike protein homotrimer that could serve as a binding site for small molecules. However, it is not known whether these molecules would inhibit, stimulate, or have no effect on viral replication. To address this, we employed structural-based screening to identify small molecules that dock into the trimer cavity and assessed their impact on viral replication. Our findings show that a cohort of identified small molecules binding to the Spike trimer cavity effectively reduces the replication of various SARS-CoV-2 variants. These molecules exhibited inhibitory effects on B.1 (European original, D614G, EDB2) and B.1.617.2 (δ) variants, while showing moderate activity against the B.1.1.7 (α) variant. We further categorized these molecules into distinct groups based on their structural similarities. Our experiments demonstrated a dose-dependent viral replication inhibitory activity of these compounds, with some, like BCC0040453 exhibiting no adverse effects on cell viability even at high concentrations. Further investigation revealed that pre-incubating virions with compounds like BCC0031216 at different temperatures significantly inhibited viral replication, suggesting their specificity towards the S protein. Overall, our study highlights the inhibitory impact of a diverse set of chemical molecules on the biological activity of the Spike protein. These findings provide valuable insights into the role of the trimer cavity in the viral replication cycle and aid drug discovery programs aimed at targeting the coronavirus family.

The association of tadalafil exposure with lower rates of major adverse cardiovascular events and mortality in a general population of men with erectile dysfunction.

BACKGROUND: Tadalafil is a long-acting phosphodiesterase-5 inhibitor (PDE-5i) indicated for erectile dysfunction (ED). HYPOTHESIS: Our hypothesis was that tadalafil will reduce the risk of major adverse cardiovascular events (MACE: composite of cardiovascular death, myocardial infarction, coronary revascularization, unstable angina, heart failure, stroke) and all-cause death in men with ED. METHODS: A retrospective observational cohort study was conducted in a large US commercial insurance claims database in men with a diagnosis of ED without prior MACE within 1 year. The exposed group (n = 8156) had ≥1 claim for tadalafil; the unexposed group (n = 21 012) had no claims for any PDE-5i. RESULTS: Primary outcome was MACE; secondary outcome was all-cause death. Groups were matched for cardiovascular risk factors, including preventive therapy. Over a mean follow-up of 37 months for the exposed group and 29 months for the unexposed group, adjusted rates of MACE were 19% lower in men exposed to tadalafil versus those unexposed to any PDE-5i (hazard ratio [HR] = 0.81; 95% confidence intervals [CI] = 0.70-0.94; p = .007). Tadalafil exposure was associated with lower adjusted rates of coronary revascularization (HR = 0.69; 95% CI = 0.52-0.90; p = .006); unstable angina (HR = 0.55; 95% CI = 0.37-0.81; p = .003); and cardiovascular-related mortality (HR = 0.45; CI = 0.22-0.93; p = .032). Overall mortality rate was 44% lower in men exposed to tadalafil (HR = 0.56; CI = 0.43-0.74; p < .001). Men in the highest quartile of tadalafil exposure had the lowest rates of MACE (HR: 0.40; 95% CI: 0.28-0.58; p < .001) compared to lowest exposure quartile. CONCLUSION: In men with ED, exposure to tadalafil was associated with significant and clinically meaningful lower rates of MACE and overall mortality.

FISH analysis reveals CDKN2A and IFNA14 co-deletion is heterogeneous and is a prominent feature of glioblastoma.

Deletion of CDKN2A occurs in 50% of glioblastomas (GBM), and IFNA locus deletion in 25%. These genes reside closely on chromosome 9. We investigated whether CDKN2A and IFNA were co-deleted within the same heterogeneous tumour and their prognostic implications. We assessed CDKN2A and IFNA14 deletions in 45 glioma samples using an in-house three-colour FISH probe. We examined the correlation between p16INK4a protein expression (via IHC) and CDKN2A deletion along with the impact of these genomic events on patient survival. FISH analyses demonstrated that grades II and III had either wildtype (wt) or amplified CDKN2A/IFNA14, whilst 44% of GBMs harboured homozygous deletions of both genes. Cores with CDKN2A homozygous deletion (n = 11) were negative for p16INK4a. Twenty p16INK4a positive samples lacked CDKN2A deletion with some of cells showing negative p16INK4a. There was heterogeneity in IFNA14/CDKN2A ploidy within each GBM. Survival analyses of primary GBMs suggested a positive association between increased p16INK4a and longer survival; this persisted when considering CDKN2A/IFNA14 status. Furthermore, wt (intact) CDKN2A/IFNA14 were found to be associated with longer survival in recurrent GBMs. Our data suggest that co-deletion of CDKN2A/IFNA14 in GBM negatively correlates with survival and CDKN2A-wt status correlated with longer survival, and with second surgery, itself a marker for improved patient outcomes.

Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry.

MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA-binding protein, translation factor, and molecular chaperone. The small molecule Nutlin-3 has been engineered to bind to the N-terminal hydrophobic pocket domain of MDM2. This binding of Nutlin-3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2-p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein-protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin-3 binding to the N-terminal domain of MDM2 (MDM2(1-126)) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin-3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55-60 exhibited slower deuterium exchange after Nutlin-3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103-107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin-3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2(Y104G) and MDM2(L107G) mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2(Y104G) complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2(Y104G) complex retained normal Nutlin-3 responsiveness. These data reveal the existence of a second functional Nutlin-3-binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2.

Strategies for clinical dose optimization of T cell-engaging therapies in oncology.

Innovative approaches in the design of T cell-engaging (TCE) molecules are ushering in a new wave of promising immunotherapies for the treatment of cancer. Their mechanism of action, which generates an in trans interaction to create a synthetic immune synapse, leads to complex and interconnected relationships between the exposure, efficacy, and toxicity of these drugs. Challenges thus arise when designing optimal clinical dose regimens for TCEs with narrow therapeutic windows, with a variety of dosing strategies being evaluated to mitigate key side effects such as cytokine release syndrome, neurotoxicity, and on-target off-tumor toxicities. This review evaluates the current approaches to dose optimization throughout the preclinical and clinical development of TCEs, along with perspectives for improvement of these strategies. Quantitative approaches used to aid the understanding of dose-exposure-response relationships are highlighted, along with opportunities to guide the rational design of next-generation TCE molecules, and optimize their dose regimens in patients.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.