Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry

Antibody drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to amonoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules afford highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination, and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analysers and more recently Orbitrap instruments. Herein we describe for the first time the use of a 15 Tesla solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodisation function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine and cysteine biotin-conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ±0.02 versus 2.24 ±0.02 for a 5-molar equivalent and 3.99 ±0.09 versus 4.43 ±0.01 for a 10-molar equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin load, when using standard ESI conditions as opposed to nanoESI native-MS conditions. Both denatured LC-MS and native-MS spectral data were deconvoluted using a parsimonious based algorithm, without the need for parameter adjustment

A phase I study of intravenous liposomal daunorubicin (DaunoXome) in paediatric patients with relapsed or resistant solid tumours

Anthracyclines are widely used in paediatric oncology, but their use is limited by the risk of cumulative cardiac toxicity. Encapsulating anthracyclines in liposomes may reduce cardiac toxicity and possibly increase drug availability to tumours. A phase I study in paediatric patients was designed to establish the dose limiting toxicity (DLT) and maximum tolerated dose (MTD) after a single course of liposomal daunorubicin, ‘DaunoXome', as a 1 h infusion on day 1 of a 21 day cycle. Patients were stratified into two groups according to prior treatment: Group A (conventional) and group B (heavily pretreated patients). Dose limiting toxicity was expected to be haematological, and a two-step escalation was planned, with and without G-CSF support. Pharmacokinetic studies were carried out in parallel. In all, 48 patients aged from 1 to 18 years were treated. Dose limiting toxicity was neutropenia for both groups. Maximum tolerated dose was defined as 155 mg m−2 for Group A and 100 mg m−2 for Group B. The second phase with G-CSF was interrupted because of evidence of cumulative cardiac toxicity. Cardiac toxicity was reported in a total of 15 patients in this study. DaunoXome shares the early cardiotoxicity of conventional anthracyclines in paediatric oncology. This study has successfully defined a haematological MTD for DaunoXome, but the significance of this is limited given the concerns of delayed cardiac toxicity. The importance of longer-term follow-up in patients enrolled into phase I studies has been underestimated previously, and may lead to an under-recognition of important adverse events

Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain

Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general

Endocrinologic, neurologic, and visual morbidity after treatment for craniopharyngioma

Craniopharyngiomas are locally aggressive tumors which typically are focused in the sellar and suprasellar region near a number of critical neural and vascular structures mediating endocrinologic, behavioral, and visual functions. The present study aims to summarize and compare the published literature regarding morbidity resulting from treatment of craniopharyngioma. We performed a comprehensive search of the published English language literature to identify studies publishing outcome data of patients undergoing surgery for craniopharyngioma. Comparisons of the rates of endocrine, vascular, neurological, and visual complications were performed using Pearson’s chi-squared test, and covariates of interest were fitted into a multivariate logistic regression model. In our data set, 540 patients underwent surgical resection of their tumor. 138 patients received biopsy alone followed by some form of radiotherapy. Mean overall follow-up for all patients in these studies was 54 ± 1.8 months. The overall rate of new endocrinopathy for all patients undergoing surgical resection of their mass was 37% (95% CI = 33–41). Patients receiving GTR had over 2.5 times the rate of developing at least one endocrinopathy compared to patients receiving STR alone or STR + XRT (52 vs. 19 vs. 20%, χ2P < 0.00001). On multivariate analysis, GTR conferred a significant increase in the risk of endocrinopathy compared to STR + XRT (OR = 3.45, 95% CI = 2.05–5.81, P < 0.00001), after controlling for study size and the presence of significant hypothalamic involvement. There was a statistical trend towards worse visual outcomes in patients receiving XRT after STR compared to GTR or STR alone (GTR = 3.5% vs. STR 2.1% vs. STR + XRT 6.4%, P = 0.11). Given the difficulty in obtaining class 1 data regarding the treatment of this tumor, this study can serve as an estimate of expected outcomes for these patients, and guide decision making until these data are available

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Aging Processes in Lithiated FeSn2 Based Negative Electrode for Li- Ion Batteries: A New Challenge for Tin Based Intermetallic Materials

Tin-based intermetallic compounds proposed as negative electrode materials for Li-ion batteries not only suffer from capacity fade during cycling due to volume variations but also from aging phenomena in lithiated states. By using FeSn2 as a model compound, we propose an analysis of this process by combining electrochemical potential measurements, 119Sn and 57Fe Mössbauer spectroscopies, magnetic measurements, and impedance spectroscopy. We show that the Fe/Li7Sn2 composite obtained at the end of the first discharge is progressively transformed during the aging process occurring within the electrochemical cell in open circuit condition. The Fe nanoparticles are stable while the Li7Sn2 nanoparticles are progressively delithiated with time leading to Sn-rich LixSn nanoalloys without observable back reaction with Fe. The deinserted lithium atoms react with the electrolyte and modify the surface electrode interphase (SEI) by increasing its thickness and/or decreasing its porosit

Splenic F-18-FDG uptake on baseline PET/CT is associated with oncological outcomes and tumor immune state in uterine cervical cancer

OBJECTIVE: The spleen represents an important contributor to tumor immune escape, but the relevance of increased splenic metabolic activity remains to be fully elucidated. METHODS: We retrospectively measured the spleen-to-liver standard uptake value (SLR) on 18F-FDG PET/CT examinations of 92 consecutive patients with FIGO stage IB1 to IVA cervical cancer and integrated the results with survival, response to treatment, tumor immune infiltrate, and baseline characteristics. RESULTS: SLRmax > 0.92 (p = .026) and SLRmean > 0.94 (p = .005) were significantly associated with decreased DFS in univariable analysis. Multivariable models were built using best subset selection; ΔSLRmax and either SLRmax or SLRmean were consistently selected, strongly reinforcing the association between SLR variables and DFS in relation to potential confounders (all models p ≤ .002). Independent associations were found for SLRmax using multivariable Cox regression models for DFS (all p ≤ .003). Further, uni- and multivariable analyses demonstrated the negative impact of higher SLR values on pathological complete response. A statistically significant higher proportion of patients with high SLRmax had a dense infiltrate of CD20+ (p = .036) and CD68+ (p = .015) immune cells, as well as PD-L1+ tumor cells (p = .019) as compared to those with low SLRmax. Finally, high SLRmax status was neither associated with systemic inflammatory markers (except for an increased white blood cell count; p = .038), nor with clinically overt infection. CONCLUSION: This hypothesis-generating study provides the first evidence that increased splenic metabolic activity is a negative prognostic and predictive biomarker in locally advanced cervical cancer. In addition, it might help to discriminate immunologically 'hot' from 'cold' cervical tumors.status: publishe

Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes

Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. Graphical Abstract ᅟ