Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through ...more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved ...digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the ...identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ∼50% of the mitochondrial proteome below 30 kDa and over 75% of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins containing between 1 and 12 transmembrane helices, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were observed, many harboring post-translational modifications, including over a dozen proteins containing lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large numbers of intact proteoforms in a high-throughput fashion.
The objective of this study was to provide an updated estimate of the effectiveness of belt-positioning booster (BPB) seats compared with seat belts alone in reducing the risk for injury for children ...aged 4 to 8 years.
Data were collected from a longitudinal study of children who were involved in crashes in 16 states and the District of Columbia from December 1, 1998, to November 30, 2007, with data collected via insurance claims records and a validated telephone survey. The study sample included children who were aged 4 to 8 years, seated in the rear rows of the vehicle, and restrained by either a seat belt or a BPB seat. Multivariable logistic regression was used to determine the odds of injury for those in BPB seats versus those in seat belts. Effects of crash direction and booster seat type were also explored.
Complete interview data were obtained on 7151 children in 6591 crashes representing an estimated 120646 children in 116503 crashes in the study population. The adjusted relative risk for injury to children in BPB seats compared with those in seat belts was 0.55.
This study reconfirms previous reports that BPB seats reduce the risk for injury in children aged 4 through 8 years. On the basis of these analyses, parents, pediatricians, and health educators should continue to recommend as best practice the use of BPB seats once a child outgrows a harness-based child restraint until he or she is at least 8 years of age.
Abstract
The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based ...multiplexed workflow for top-down tandem MS (MS
2
) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS
2
identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.
Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, ...limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data. Combining mass determination via spectral deconvolution with a top-down database search and stoichiometry calculations, ProSight Native can determine the complete composition of protein complexes. To demonstrate its features, we used ProSight Native to successfully determine the composition of the homotetrameric membrane complex Aquaporin Z. We also revisited previously published spectra and were able to decipher the composition of a heterodimer complex bound with two noncovalently associated ligands. In addition to determining complex composition, we developed new tools in the software for validating native mass spectrometry fragment ions and mapping top-down fragmentation data onto three-dimensional protein structures. Taken together, ProSight Native will reduce the informatics burden on the growing field of native mass spectrometry, enabling the technology to further its reach.
Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced ...volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo 'features' such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0–30 kDa mass range extracted from whole cell or tissue lysates. ...Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography–tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
Currently, many states are upgrading their child restraint laws to include provisions for the use of age-appropriate restraints through 6 to 8 years of age, with some also requiring rear seating for ...children, enabling the laws to be in closer alignment with best-practice recommendations.
To evaluate the relationships of seating position and restraint status to the risk of injury among children in passenger vehicle crashes.
This was a cross-sectional study of children <16 years of age who were involved in crashes of insured vehicles in 15 states, with data collected via insurance claims records and a telephone survey. A probability sample of 17980 children in 11506 crashes, representing 229106 children in 146613 crashes, was collected between December 1, 1998, and November 30, 2002. Parent reports were used to define restraint status, seating position, and occurrence of clinically significant injuries, with the use of a previously validated instrument.
Approximately 62% of the children used seat belts, 35% used child restraints, and 3% used no restraint. Nearly 4 of 5 children sat in the rear seat, with one half of all children being restrained appropriately for their age in the rear, although this varied according to the age of the child. Overall, 1.6% of children suffered serious injuries, 13.5% had minor injuries, and 84.9% did not have any injury. Unrestrained children in the front were at the highest risk of injury and appropriately restrained children in the rear were at the lowest risk, for all age groups. Inappropriately restrained children were at nearly twice the risk of injury, compared with appropriately restrained children (odds ratio OR: 1.8; 95% confidence interval CI: 1.4-2.3), whereas unrestrained children were at >3 times the risk (OR: 3.2; 95% CI: 2.5-4.1). The effect of seating row was smaller than the effect of restraint status; children in the front seat were at 40% greater risk of injury, compared with children in the rear seat (OR: 1.4; 95% CI: 1.2-1.7). Had all children in the study population been appropriately restrained in the rear seat, 1014 serious injuries (95% CI: 675-1353 injuries) would have been prevented (with the assumption that restraint effectiveness does not depend on a variety of other driver-related, child-related, crash-related, vehicle-related, and environmental factors).
Age-appropriate restraint confers relatively more safety benefit than rear seating, but the 2 work synergistically to provide the best protection for children in crashes. These results support the current focus on age-appropriate restraint in recently upgraded state child restraint laws. However, it is important to note that considerable added benefit would be realized with additional requirements for rear seating.
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