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The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.
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Antineoplásicos , Inmunoconjugados , Ratas , Animales , Humanos , Ligasas , Inmunoconjugados/farmacología , Antineoplásicos/farmacología , Células MCF-7 , Trastuzumab/farmacología , Línea Celular TumoralRESUMEN
Durvalumab has been administered to patients with unresectable stage III non-small cell lung cancer (NSCLC). However, it remains unclear whether durvalumab benefits these patients with epidermal growth factor receptor (EGFR) mutation. We conducted a retrospective, multicenter study of patients with EGFR mutation who received chemoradiotherapy (CRT) between June 2018 and March 2021. We assessed patient characteristics, efficacy of durvalumab, and durvalumab safety before and after targeted therapy. We collected data on a total of 673 patients, of whom 401 (59.6%) underwent EGFR mutation testing. Fifty-one patients were EGFR positive and 311 were EGFR negative. In the EGFR-positive group, there were higher proportions of females, never-smokers, and patients with adenocarcinoma histology. Of the 51 patients in the positive group and 311 in the negative group who received CRT, 45 (88.2%) and 247 (79.4%) received durvalumab, with median progression-free survival of 23.0 and 24.2 months in the positive and negative groups, respectively (hazard ratio 1.03; 95% confidence interval: 0.64-1.67). The main adverse event was pneumonitis (positive group: 62.2%; 4.4% grade 3; negative group: 62.3%; 6.9% grade 3). No treatment-related deaths were observed. Of the 45 patients in the positive group who received durvalumab, 14 (31.1%) received targeted therapy after durvalumab at the data cutoff. One patient discontinued targeted therapy after developing pneumonitis. In patients with unresectable stage III NSCLC with EGFR mutation, durvalumab after CRT is potentially safe and effective. This may be a suitable treatment sequence for these patients.
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Anticuerpos Monoclonales , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neumonía , Femenino , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Estudios Retrospectivos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Quimioradioterapia , Mutación , Receptores ErbB/genéticaRESUMEN
The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptorâ 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000â times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.
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Péptidos , Factor A de Crecimiento Endotelial Vascular , Péptidos/química , Péptidos y Proteínas de Señalización IntercelularRESUMEN
Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.
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Aminoácidos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Aminoácidos/química , Anticuerpos Monoclonales/química , Reacciones Antígeno-Anticuerpo , Antígenos/química , Sitios de Unión , Halogenación , Modelos MolecularesRESUMEN
Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.
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Factor de Crecimiento de Hepatocito/análisis , Compuestos Macrocíclicos/química , Neoplasias Experimentales/diagnóstico por imagen , Imagen Óptica , Péptidos/química , Animales , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/metabolismo , Compuestos Macrocíclicos/farmacología , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Péptidos/farmacología , Tomografía de Emisión de PositronesRESUMEN
Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.
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Ácidos Grasos/química , Lípidos/química , Nanopartículas/química , Polietilenglicoles/química , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Molécula de Adhesión Celular Epitelial/metabolismo , Femenino , Silenciador del Gen/efectos de los fármacos , Células HCT116 , Humanos , Ligandos , Ratones , Ratones Endogámicos ICR , Ratones SCID , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Péptidos/química , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
Natural killer (NK) cells play an important role in tumor immunity and infection control. The natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 are involved in the control of the activation of NK cells. Few reports have investigated the ligands of NCRs. We previously reported the NCRs binding affinity to heparin and glycosaminoglycans. We also showed that multimeric sialyl Lewis X-expressing transferrin, secreted by human hepatoma HepG2 cells, binds to NKp46 and NKp44, but not to NKp30. In this study, we investigated the binding between NCRs and glycolipids. The possible binding of glycolipids to NCRs was screened by microarray, using the recombinant extracellular domain of NKp46, NKp44 and NKp30 tagged with 6×His (rNKp46, rNKp44 and rNKp30). We found that rNKp44 binds to Globo-A. However, we did not detect the interaction between rNKp46 or rNKp30 and any of the glycolipids investigated. Direct binding assays supported the results of the microarray screening. Therefore, we concluded that Globo-A is a novel ligand for NKp44 but not NKp46 and NKp30, and showed differences in the ligand selectivity of NCRs.
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Glucolípidos/metabolismo , Receptores Gatillantes de la Citotoxidad Natural/metabolismo , Línea Celular , Humanos , Ligandos , Análisis por Micromatrices , Proteínas Recombinantes/metabolismoRESUMEN
Non-native ligands for growth factor receptors with distinct chemical properties and different biological activities have the potential to become therapeutic applications. We previously generated MET/hepatocyte growth factor (HGF) receptor agonists using bivalent macrocyclic peptides. The highest MET-activating agonists exhibited biological activity that was indistinguishable from the effects of HGF. In this study, we investigated MET activation, signal characteristics, and biological responses induced by a macrocyclic peptide partial agonist known as aML5-PEG11. aML5-PEG11 induced weak tyrosine phosphorylation of MET while enhancing cell migration with potency comparable to HGF. aML5-PEG11 induced marked AKT (protein kinase B) and ERK (extracellular signal-regulated kinase) activation at a comparable potency and time-dependency to HGF, which suggests that enhancement of cell motility is attributable to activation of these molecules. In a 3-D culture of bile duct cancer cells in collagen gel, HGF induced robust activation of MET, ERK, and AKT, which was associated with enhanced expression of genes involved in bile duct development and subsequent branching of tubulogenesis. In contrast, aML5-PEG11 induced marginal activation of MET, ERK, and AKT (levels near the detection limits), which was associated with failure to enhance the expression of genes involved in bile duct development and a lack of tubulogenic response. Thus, MET activation by aML5-PEG11 couples to biological responses differently from HGF in an extracellular context-dependent manner.
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Factor de Crecimiento de Hepatocito/metabolismo , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Activación Transcripcional , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Péptidos/química , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-met/agonistasRESUMEN
The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibits the cellular uptake of LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use in an innovative nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard peptides integrated discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for delivering therapeutics were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.
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Sistemas de Liberación de Medicamentos , Molécula de Adhesión Celular Epitelial/genética , ARN Interferente Pequeño/farmacología , Animales , Péptidos Catiónicos Antimicrobianos , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial/metabolismo , Proteínas de Peces , Técnicas de Silenciamiento del Gen , Humanos , Lípidos/química , Liposomas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Nanopartículas/química , Tamaño de la Partícula , Polietilenglicoles/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aim of this study was to examine the feasibility of short volume hydration (SH) with magnesium and mannitol versus normal high volume hydration (NH) for targeting nephrotoxicity in lung cancer patients treated with cisplatin (CDDP)- containing chemotherapy. Between January 2012 and February 2013, 28 patients with lung cancer at a single institute received CDDP-containing chemotherapy. We retrospectively evaluated the incidence of nephrotoxicity during the first cycle of chemotherapy. Nephrotoxicity was compared between the SH and NH regimens according to the Common Terminology Criteria for Adverse Events(CTCAE) version 4.0. Laboratory data were collected from the 2 regimen groups at pre-treatment, during the first cycle, and post-treatment and were compared by univariate analysis. Twelve patients received the SH regimen with magnesium and mannitol, and 16 patients received the NH regimen. Only 1 patient in the NH regimen group had Grade 2 increases in serum creatinine. On the other hand, no patient in the SH regimen group had increased serum creatinine. There was no significant difference in the incidence of nephrotoxicity between the 2 regimen groups during the first cycle of CDDP induction(p=0.38). The SH regimen with magnesium and mannitol is feasible in lung cancer patients treated with CDDP-containing chemotherapy.
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Cisplatino/efectos adversos , Enfermedades Renales/terapia , Neoplasias Pulmonares/tratamiento farmacológico , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , Estudios de Factibilidad , Femenino , Fluidoterapia , Humanos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/prevención & control , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
OBJECTIVE: This study aimed to investigate the feasibility and validity of measuring implicit attitudes towards dementia in adults and older adults and evaluate the impact of dementia-friendly education using virtual reality (VR) on implicit attitudes. METHODS: This study was a secondary analysis of data from a randomised controlled trial. Community members in Tokyo aged 20-90 years participated in dementia-friendly education with or without VR. At the end of the dementia-friendly education programs, implicit attitudes towards dementia were measured using the Implicit Relational Assessment Procedure (IRAP). RESULTS: Of the 145 participants, 89 (61%) started the IRAP, and 21 (15%) completed it. Lower age was significantly associated with the start/completion of the IRAP, and the age thresholds at which 50% of participants would not start/complete it were estimated to be 72.3/44.8 years, respectively. Those who had experience interacting with people with dementia other than family members had lower IRAP scores than those who had no such experience. The intervention group participating in the VR program had lower IRAP scores than the control group (p = .09). CONCLUSIONS: Although measuring implicit attitudes using IRAP is deemed not feasible for people in their 70s and older, the differences in interaction experience would be evidence supporting the validity of the measurements of implicit attitudes towards dementia. The results suggest that dementia-friendly education, using VR, improves implicit attitudes towards dementia.
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BACKGROUND AND OBJECTIVES: Raising dementia awareness is essential for building a dementia-friendly community. However, existing studies have underexplored the effects of virtual reality (VR) dementia educational programs for the general public on enhancing positive attitude toward dementia. This study aimed to examine the effectiveness of a VR dementia-friendly educational program called the Drive for Dementia Readiness Inside Virtual Reality (DRIVE) program to improve attitude toward dementia of the general public. RESEARCH DESIGN AND METHODS: A two-arm randomized controlled trial was conducted. Eligibility criteria for participants included being 16 years old and above in Japan and having no professional license in healthcare and social care. We randomized individuals to attend a multi-element VR dementia-friendly educational program (intervention), including simulation, VR films, short films, lectures, and discussions or a lecture-based program (control). Data were collected three times, including at baseline, post-intervention, and 3-month follow-up. The primary outcome was attitude toward dementia. The secondary outcomes were intention of helping behavior for people living with dementia and knowledge of dementia. RESULTS: We recruited 157 community residents, among whom 130 were included in the analysis. Although the mean changes in attitude score were not significantly different between the groups (Hedge's g = .26), the intention of helping behavior score was significantly higher in the intervention group (g = .49). DISCUSSION AND IMPLICATION: The DRIVE, a VR-based multi-element dementia-friendly educational intervention, was shown as a promising tool for significantly impacting the intention of helping behavior for people living with dementia to establish dementia-friendly communities.
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BACKGROUND: Selecting pembrolizumab monotherapy (MONO) or pembrolizumab plus platinum-based chemotherapy (COMB) for patients with nonsmall cell lung cancer (NSCLC) and high programmed death-ligand 1 (PD-L1) expression is an important issue in clinical practice. We previously conducted a retrospective multicenter observational study of patients with NSCLC and high PD-L1 expression who received MONO or COMB as a first-line treatment. Here, we report updated data and evaluate the long-term outcomes. METHODS: We performed a retrospective multicenter study of 298 patients with NSCLC and high PD-L1 expression who received MONO or COMB as first-line treatment between December 2018 and January 2020. We reviewed the medical records and assessed the clinical efficacy and toxicity using a prolonged data cutoff. RESULTS: In total, 164 (median age: 74 years) and 134 (median age: 68 years) patients received MONO and COMB, respectively; patients who received COMB were younger and had better performance statuses (0-1). At the prolonged data cutoff, the median follow-up was 20.2 (range: 0.1-41.4) months. The median progression-free survivals were 7.5 and 13.1 months, and overall survivals (OSs) were 17.2 and 33.7 months for MONO and COMB, respectively. Treatment discontinuation rates were 21.9% and 20.1% for the MONO and COMB, respectively. With prolonged follow-up, although COMB demonstrated an OS benefit and higher objective response rate than MONO, in the propensity score matching analysis COMB didn't demonstrate a significant benefit compared to the MONO. CONCLUSIONS: COMB may be effective as a first-line treatment for NSCLC with high PD-L1 expression in a selected subset of patients.
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Anticuerpos Monoclonales Humanizados , Antígeno B7-H1 , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Masculino , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/metabolismo , Femenino , Anciano , Antígeno B7-H1/metabolismo , Estudios Retrospectivos , Persona de Mediana Edad , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Adulto , Resultado del Tratamiento , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.
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ADN/química , Oligonucleótidos/química , ARN/química , Telómero/química , ADN/metabolismo , Humanos , Oligonucleótidos/metabolismo , ARN/metabolismo , Telómero/metabolismoRESUMEN
Amrubicin, a third-generation synthetic anthracycline agent, has favorable clinical activity and acceptable toxicity for the treatment of patients with non-small cell lung cancer (NSCLC) and small cell lung cancer. We conducted this study to evaluate the efficacy and safety of amrubicin for advanced NSCLC patients as a third- or fourth-line therapy. Eligible patients had recurrent or refractory advanced NSCLC after second- or third-line therapy. Patients received amrubicin, 35 mg/m(2) i.v. on days 1-3 every 3 weeks. The primary endpoint was the disease control rate (DCR). Secondary endpoints were the overall survival (OS) time, progression-free survival (PFS) time, response rate, and toxicity profile. Of the 41 patients enrolled, 26 received amrubicin as a third-line and 15 received it as a fourth-line therapy. The median number of treatment cycles was two (range, 1-9). Objective responses were complete response (n = 0), partial response (n = 4), stable disease (n = 21), progressive disease (n = 15), and not evaluable (n = 1), resulting in a DCR of 61.0% (95% confidence interval, 46.0%-75.9%). The overall response rate was 9.8% (95% confidence interval, 0.6%-18.8%). The median PFS interval was 3.0 months, median OS time was 12.6 months, and 1-year survival rate was 53.7%. Grade 3 or 4 hematological toxicities were neutropenia (68%), anemia (12%), thrombocytopenia (12%), and febrile neutropenia (17%). Nonhematological toxicities were mild and reversible. No treatment-related deaths were observed. Amrubicin showed significant clinical activity with manageable toxicities as a third- or fourth-line therapy for patients with advanced NSCLC. This study provides relevant data for routine practice and future prospective trials evaluating third- or fourth-line treatment strategies for patients with advanced NSCLC.
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Antraciclinas/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Adulto , Anciano , Antraciclinas/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Terapia Combinada , Supervivencia sin Enfermedad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de NeoplasiasRESUMEN
Telomeric repeat-containing RNA (referred to as TERRA), a noncoding RNA molecule, has recently been found in mammalian cells. The detailed structural features and function of the TERRA RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In the present studies, we investigated the structural features of human TERRA RNA in living cells. Using a light-switching pyrene probe, we found that human TERRA RNA forms a parallel G-quadruplex structure in living cells, providing the in vivo evidence for the presence of the G-quadruplex in human TERRA RNA. Furthermore, imaging experiments clearly show that TERRA RNA G-quadruplex localizes to telomere DNA at cell nuclei. These results provide valuable information to allow understanding of the structure and function of human TERRA RNA.
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G-Cuádruplex , ARN no Traducido/química , Secuencias Repetitivas de Ácidos Nucleicos/genética , Telómero/genética , Secuencia de Bases , Dicroismo Circular , Colorantes Fluorescentes/química , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Pirenos/química , ARN no Traducido/genética , Espectrometría de FluorescenciaRESUMEN
In this review, we discuss emerging technologies for drug discovery, which yields novel molecular scaffolds based on natural product-inspired non-traditional peptides expressed using the translation machinery. Unlike natural products, these technologies allow for constructing mRNA-encoding libraries of macrocyclic peptides containing non-canonical sidechains and N-methyl-modified backbones. The complexity of sequence space in such libraries reaches as high as a trillion (>1012), affording initial hits of high affinity ligands against protein targets. Although this article comprehensively covers several related technologies, we discuss in greater detail the technical development and advantages of the Random non-standard Peptide Integration Discovery (RaPID) system, including the recent identification of inhibitors against various therapeutic targets.
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Descubrimiento de Drogas/métodos , Biblioteca de Genes , Péptidos Cíclicos/farmacología , ARN Mensajero/genética , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Código Genético , Humanos , Datos de Secuencia Molecular , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , ARN Catalítico/química , Transcripción GenéticaRESUMEN
8-Azidoadenosine 3',5'-cyclic monophosphate (8-azido cAMP) was directly detected in living cells, by applying Cu-free azide-alkyne cycloaddition to probe cAMP derivatives by fluorescence light-up. Fluorescence emission was generated by two non-fluorescent molecules, 8-azido cAMP as a model target and difluorinated cyclooctyne (DIFO) reagent as a probe. The azide-alkyne cycloaddition reaction between 8-azido cAMP and DIFO induces fluorescence in 8-azido cAMP. The fluorescence emission serves as a way to probe 8-azido cAMP in cells.
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Azidas/química , AMP Cíclico/análogos & derivados , Colorantes Fluorescentes/química , Azidas/metabolismo , Química Clic , AMP Cíclico/química , AMP Cíclico/metabolismo , Reacción de Cicloadición , Ciclooctanos/química , Ciclooctanos/metabolismo , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Análisis de la Célula Individual/métodos , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Coloración y EtiquetadoRESUMEN
Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.
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PURPOSE: Immune-checkpoint inhibitors (ICIs) are effective against advanced non-small cell lung cancer (NSCLC). However, whether the efficacy and safety of ICI treatment in elderly patients are similar to those in younger patients is unclear. This study was designed to address this question. METHODS: We enrolled patients who received ICI monotherapy in Japan between December 2015 and December 2017; those ≥75 years of age comprised the elderly group. We compared the efficacy and safety of ICI monotherapy in elderly patients with those in younger patients and explored prognostic factors in elderly patients. RESULTS: We enrolled 676 patients; 137 (20.3%) were assigned to the elderly group. The median age of the elderly and younger groups was 78 (range, 75-85) and 66 (range, 34-74) years. The median progression-free survival (4.8 months vs. 3.3 months, p = 0.1589) and median overall survival (12.3 months vs. 13.0 months, p = 0.5587) were similar between the elderly and younger groups. Multivariate analysis revealed that a significantly better OS in the elderly group was associated with better responses to first- or second-line ICI treatment (p = 0.011) and more immune-related adverse events (irAEs) (p = 0.02). IrAEs that led to ICI discontinuation occurred in 34 of 137 patients (24.8%) in the elderly group, and their survival was significantly higher than that in those who did not have irAEs. CONCLUSION: ICI is also effective in elderly NSCLC patients, and treatment discontinuation due to irAEs may be a good prognostic marker.