Sarah Cianférani

Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment

Antibody drug conjugates (ADCs) are macromolecules composed of cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). ADCs are among the most promising next generation of empowered mAbs foreseen to treat cancers. Compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. An increasing need underlying ADCs development and optimization is to improve its analytical and bioanalytical characterization by assessing three main ADC quality attributes: drug distribution, amount of naked antibody, and average drug to antibody ratio (DAR). Here, the analytical potential of native mass spectrometry (MS) and native ion mobility MS (IM-MS) is compared to hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs. Brentuximab vedotin, first in class and gold standard, was chosen for a proof of principle. High resolution native MS provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native MS for simultaneous detection of mixtures of covalent and noncovalent products. Native IM-MS was next used for the first time to characterize an ADC. IM-MS evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of IM-MS data was developed to directly extrapolate average DAR and DAR distribution. Additionally, HIC fractions were collected and analyzed by native MS and IM-MS, assessing the interpretation of each HIC peak. Altogether, our results illustrate how native MS and IM-MS can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into MS workflows for in-depth ADC analytical characterization.

Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies

The approval process for antibody biosimilars relies primarily on comprehensive analytical data to establish comparability and high similarity with the originator. Mass spectrometry (MS) in combination with liquid chromatography (LC) and electrophoretic methods are the corner stone for comparability and biosimilarity evaluation. In this special feature we report head-to-head comparison of trastuzumab and cetuximab with corresponding biosimilar and biobetter candidates based on cutting-edge mass spectrometry techniques such as native MS and ion-mobility MS at different levels (top, middle and bottom). In addition, we discuss the advantages and the limitations of sample preparation and enzymatic digestion, middle-up and -down strategies and the use of hydrogen/deuterium exchange followed by MS (HDX-MS). Last but not least, emerging separation methods combined to MS such as capillary zone electrophoresis-tandem MS (CESI-MS/MS), electron transfer dissociation (ETD), top down-sequencing (TDS) and high-resolution MS (HR-MS) that complete the panel of state-of-the-art MS-based options for comparability and biosimilarity evaluation are presented.

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine‐linked antibody drug conjugate

Antibody–drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM‐MS) for the characterization of trastuzumab emtansine (T‐DM1, Kadcyla®). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)‐positive breast cancer, and combines the anti‐HER2 antibody trastuzumab (Herceptin®), with the cytotoxic microtubule‐inhibiting maytansine derivative, DM1. We show that native MS combined with high‐resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM‐MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T‐DM1.

Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific” Monoclonal Antibody Formation

Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

Computational and Mass-Spectrometry-Based Workflow for the Discovery and Validation of Missing Human Proteins: Application to Chromosomes 2 and 14.

In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last 4 years, the French proteomics infrastructure has collected high-quality data sets from 40 human samples, including a series of rarely studied cell lines, tissue types, and sample preparations. Here we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these data sets. Screening database search results (85,326 dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-09-19). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level false discovery rate calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from four of the missing proteins detected in tissue/cell samples, which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for three of the proteins. For two of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.

Molecular responses of alveolar epithelial A549 cells to chronic exposure to titanium dioxide nanoparticles: A proteomic view.

UNLABELLED Although the biological effects of titanium dioxide nanoparticles (TiO2-NPs) have been studied for more than two decades, the mechanisms governing their toxicity are still unclear. We applied 2D-gel proteomics analysis on A549 epithelial alveolar cells chronically exposed for 2months to 2.5 or 50μg/mL of deeply characterized TiO2-NPs, in order to obtain comprehensive molecular responses that may reflect functional outcomes. We show that exposure to TiO2-NPs impacts the abundance of 30 protein species, corresponding to 22 gene products. These proteins are involved in glucose metabolism, trafficking, gene expression, mitochondrial function, proteasome activity and DNA damage response. Besides, our results suggest that p53 pathway is activated, slowing down cell cycle progression and reducing cell proliferation rate. Moreover, we report increased content of chaperones-related proteins, which suggests homeostasis re-establishment. Finally, our results highlight that chronic exposure to TiO2-NPs affects the same cellular functions as acute exposure to TiO2-NPs, although lower exposure concentrations and longer exposure times induce more intense cellular response. BIOLOGICAL SIGNIFICANCE Our results make possible the identification of new mechanisms that explain TiO2-NP toxicity upon long-term, in vitro exposure of A549 cells. It is the first article describing -omics results obtained with this experimental strategy. We show that this long-term exposure modifies the cellular content of proteins involved in functions including mitochondrial activity, intra- and extracellular trafficking, proteasome activity, glucose metabolism, and gene expression. Moreover we observe modification of content of proteins that activate the p53 pathway, which suggest the induction of a DNA damage response. Technically, our results show that exposure of A549 cells to a high concentration of TiO2-NPs leads to the identification of modulations of the same functional categories than exposure to low, more realistic concentrations. Still the intensity differs between these two exposure scenarios. We also show that chronic exposure to TiO2-NPs induces the modulation of cellular functions that have already been reported in the literature as being impacted in acute exposure scenarios. This proves that the exposure protocol in in vitro experiments related to nanoparticle toxicology might be cautiously chosen since inappropriate scenario may lead to inappropriate and/or incomplete conclusions.

CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability.

Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.

Comparative Expression Study of the Endo–G Protein Coupled Receptor (GPCR) Repertoire in Human Glioblastoma Cancer Stem-like Cells, U87-MG Cells and Non Malignant Cells of Neural Origin Unveils New Potential Therapeutic Targets

Glioblastomas (GBMs) are highly aggressive, invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these, cells endowed with stem properties, tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells, termed cancer stem-like cells, have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs), a family of membrane receptors, play a prominent role in cell signaling, cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here, we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs), U-87 MG cells, human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated, 138 were retained for comparative studies between the different cell types. At the transcriptomic level, eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets.

TCTP contains a BH3-like domain, which instead of inhibiting, activates Bcl-xL.

Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members’ structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11–31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP+/− mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.

A combined proteomic and targeted analysis unravels new toxic mechanisms for zinc oxide nanoparticles in macrophages.

UNLABELLED The cellular responses of the J774 macrophage cell line to zinc oxide and zirconium oxide nanoparticles have been studied by a comparative quantitative, protein level based proteomic approach. The most prominent results have been validated by targeted approaches. These approaches have been carried out under culture conditions that stimulate mildly the aryl hydrocarbon receptor, thereby mimicking conditions that can be encountered in vivo in complex environments. The comparative approach with two nanoparticles allows to separate the common responses, which can be attributed to the phagocytosis event per se, from the response specific to each type of nanoparticles. The zinc-specific responses are the most prominent ones and include mitochondrial proteins too, but also signaling molecules such as MyD88, proteins associated with methylglyoxal detoxification (glyoxalase 2, aldose reductase) and deoxyribonucleotide hydrolases. The in cellulo inhibition of GAPDH by zinc was also documented, representing a possible source of methylglyoxal in the cells, leading to an increase in methylglyoxal-modified DNA bases. These observations may be mechanistically associated with the genotoxic effect of zinc and its selective effects on cancer cells. BIOLOGICAL SIGNIFICANCE The responses of the murine J774 macrophage cell lines to two types of metallic oxide nanoparticles (zinc oxide and zirconium dioxide) were studied by a comparative 2D gel based approach. This allows sorting of shared responses from nanoparticle-specific responses. Zinc oxide nanoparticles induced specifically a strong decrease in the mitochondrial function, in phagocytosis and also an increase in the methylglyoxal-associated DNA damage, which may explain the well known genotoxicity of zinc. In conclusion, this study allows highlighting of pathways that may play an important role in the toxicity of the zinc oxide nanoparticles.