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Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.
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Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , Anticuerpos Monoclonales/química , Cromatografía en Gel , Espectrometría de Masas/métodosRESUMEN
Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(ß-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.
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Asparaginasa/química , Cromatografía Liquida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Glucosamina/química , Espectrometría de Masas en Tándem/métodos , Anticuerpos Monoclonales/metabolismo , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. METHOD: We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. RESULTS: 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized ß1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnß1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. CONCLUSIONS: This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.
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Células Endoteliales , Integrinas , Molécula de Adhesión Celular del Leucocito Activado , Animales , Anticuerpos/metabolismo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Integrina beta1/metabolismo , Integrinas/metabolismo , RatonesRESUMEN
KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.
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Antineoplásicos , Neoplasias , Acetonitrilos/uso terapéutico , Antineoplásicos/uso terapéutico , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Piperazinas , Proteínas Proto-Oncogénicas p21(ras)/genética , PirimidinasRESUMEN
Antibody-drug conjugates (ADCs) represent an important class of new biopharmaceutical modalities. ADCs are highly complex and heterogeneous molecules, potentially containing numerous product-related structures, that can contribute to the quality, efficacy and safety of the product. To keep up with product life cycle related changes, wide-range and targeted characterization of product quality attributes (PQA) are of high demand. Multi-attribute methods (MAM) can screen numerous PQAs in a parallel fashion including product properties as well as product and process-related impurities. MAM is usually based on a bottom-up approach relying on the enzymatic digestion of the protein into peptides prior to mass spectrometry (MS). However, this processing workflow can result in considerable information loss, such as the drug distribution profile of an antibody-drug conjugate. Therefore, complementary MAM approaches, based on subunit and intact mass analyses, are necessary approaches offering the advantage of product identity confirmation, quantification of the different conjugated species and monitoring the drug-to-antibody ratio at the same time. In this work we introduce a high throughput MS based attribute tracking method for ADC characterization at the intact and subunit levels by simultaneously monitoring multiple PQAs. The workflow includes sample preparation and MS instrument suitability testing for heterogeneous lysine-linked ADCs, software solutions for routine PQAs tracking, method repeatability and an easy data review fitting perfectly into high throughput analyses. As methionine oxidation is one of the modifications that should be closely monitored at any step of process development, an important application to oxidative stress evaluation using forced degradation demonstrated the applicability of the workflow.
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Productos Biológicos , Inmunoconjugados , Anticuerpos , Espectrometría de Masas , Proyectos de InvestigaciónRESUMEN
The long-term impact of persistent pulmonary vascular obstruction after pulmonary embolism (PE) remains unknown. Based on ventilation-perfusion lung scan performed at discharge and 3 months after a first PE, we aimed to investigate the prognostic value on 5-year adverse events of (1) residual pulmonary vascular obstruction (RPVO) at discharge (DIS-RPVO), (2) RPVO at 3 months (3M-RPVO), and (3) relative change in RPVO between the 2 scans (RC-RPVO). We performed a prospective, multicenter cohort study from January 2007 to December 2009 including patients who survived at least 3 months after a PE. RC-RPVO was defined as (DIS-RPVO - 3M-RPVO)/DIS-RPVO. The primary end point was a combined end point at 5 years, composed of all-cause death, recurrent venous thromboembolism, chronic thromboembolic pulmonary hypertension, heart failure, and rehospitalization for cardiac causes. Receiver-operating characteristic curves were computed to define thresholds of DIS-RPVO, 3M-RPVO, and RC-RPVO predictive of the primary combined end point at 5 years. Overall, 241 patients were included (high-risk PE: 11.2%, intermediate-risk PE: 51.8%, low-risk PE: 37%). Mean DIS-RPVO was 27.9 ± 15.1%, mean 3M-RPVO was 10.3 ± 10.8%, and mean RC-RPVO was 61.7 ± 33.4%. At 5 years, 112 patients (46.5%) experienced the combined end point. Both 3M-RPVO ≥15% and RC-RPVO ≤37.5% were independently related to the occurrence of the combined end point at 5 years (p = 0.01 and p = 0.02, respectively). DIS-RPVO did not predict long-term adverse events. In conclusion, RC-RPVO ≤37.5% and 3M-RPVO ≥15% were independently related to the occurrence of adverse events 5 years after a first PE.
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Arteriopatías Oclusivas/diagnóstico , Pulmón/diagnóstico por imagen , Arteria Pulmonar , Embolia Pulmonar/complicaciones , Anciano , Arteriopatías Oclusivas/epidemiología , Arteriopatías Oclusivas/etiología , Angiografía por Tomografía Computarizada , Femenino , Estudios de Seguimiento , Francia/epidemiología , Humanos , Masculino , Morbilidad/tendencias , Pronóstico , Estudios Prospectivos , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/mortalidad , Tasa de Supervivencia/tendencias , Factores de TiempoRESUMEN
INTRODUCTION: Gemcitabine is a chemotherapeutic agent frequently used by for the treatment of several malignancies both in the adjuvant and metastatic setting. Although myelosuppression is the most adverse event of this therapy, gemcitabine might induce severe pulmonary toxicities. We describe a case of pulmonary veno-occlusive disease (PVOD) related to gemcitabine. CASE PRESENTATION: The patient was an 83-year-old man with a metastatic pancreatic cancer who was treated by gemcitabine as first-line therapy. He was in good health and received no other chemotherapy. A dose of 1000 mg/m(2) of gemcitabine was administered over a 30-minute intravenous infusion on days 1, 8, and 15 of a 28-day cycle. After a period of 6 months, a complete response was observed. Nevertheless, the patient developed a severe dyspnea, with arterial hypoxemia and very low lung diffusion for carbon monoxide. A CT scan showed diffuse ground glass opacities with septal lines, bilateral pleural effusion, and lymph node enlargement. On echocardiography, there was a suspicion of pulmonary hypertension with elevated systolic pulmonary artery pressure and normal left ventricular pressures. Right heart catheterization confirmed pulmonary hypertension and normal pulmonary artery occlusion pressure. Diagnosis of PVOD was made, and a gemcitabine-induced toxicity was suspected. A symptomatic treatment was started. At last follow-up, patient was in functional class I with near-normal of CT scan, arterial blood gases, and echocardiography. A gemcitabine-induced PVOD is the more likely diagnosis.
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Clofibrate is a peroxisome proliferator known to induce liver tumours in rats. A proteomics study was conducted to provide new insights into the molecular mechanisms of clofibrate-induced non-genotoxic hepatocarcinogenesis. Rats were treated with 250 mg/kg day clofibrate orally and sacrificed after 7 days. Proteins extracted from the liver were analysed by 2-DE using DIGE technology. The protein identification performed by MS showed that clofibrate induced up-regulation of 77 proteins and down-regulation of 27 proteins. The highest expression ratios corresponded to proteins involved in a series of biochemical pathways such as lipid metabolism, fatty acid metabolism, amino acid metabolism, protein metabolism, citric acid cycle, xenobiotic detoxification and oxidative stress. Proteins implicated in cell proliferation and apoptosis, such as prohibitin, 10-formyl tetrahydrofolate dehydrogenase, senescence marker protein-30, pyridoxine 5'-phosphate oxidase and vimentin, were also identified as being regulated. These results provide leads for further investigations into the molecular mechanisms of liver tumours induced by clofibrate. In addition, MS results showed that a series of regulated proteins were detected as several spots corresponding to different pI and/or M(r). Differential effects on those variants could result from specific PTM and could be a specific molecular signature of the clofibrate-induced protein expression modulation in rat liver.