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[This corrects the article DOI: 10.1371/journal.pone.0234268.].
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Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.
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Anexina A1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Regulação para Cima , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/irrigação sanguínea , CamundongosRESUMO
Efficacious use of therapeutic gene delivery via nanoparticles is hampered by the challenges associated with targeted delivery to tissues of interest. Systemic administration of lipid nanoparticle (LNP)-encapsulated mRNA leads to a protein expressed predominantly in the liver and spleen. Here, LNP encapsulating mRNA was covalently conjugated to an antibody, specifically binding plasmalemma vesicle-associated protein (PV1) as a means to target lung tissue. Systemic administration of PV1-targeted LNPs demonstrated significantly increased delivery of mRNA to the lungs and a 40-fold improvement in protein expression in the lungs, compared with control LNPs. We also investigated the effect of LNP size to determine optimal tissue distribution and transfection. Larger-size PV1-targeted LNPs not only have the elasticity to target the PV1 expressed in the caveolae but also enable robust mRNA expression in the lungs. Targeted delivery of mRNA to the lungs is a promising approach in the treatment of lung diseases.
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Portadores de Fármacos/química , Lipídeos/química , Pulmão/metabolismo , Nanopartículas/química , RNA Mensageiro/farmacologia , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Cavéolas/imunologia , Feminino , Técnicas de Transferência de Genes , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB CRESUMO
Targeted strategies to deliver and retain drugs to kidneys are needed to improve drug accumulation and efficacy in a myriad of kidney diseases. These drug delivery systems show potential for improving the therapeutic windows of drugs acting in the kidney. Biodistribution of antibody-based therapeutics in vivo is governed by several factors including binding affinity, size, and valency. Investigations of how the biophysical and biochemical properties of biologics enable them to overcome biological barriers and reach kidneys are therefore of interest. Although renal accumulation of antibody fragments in cancer diagnostics and treatment has been observed, reports on effective delivery of antibody fragments to the kidneys remain scarce. Previously, we demonstrated that targeting plasmalemma vesicle-associated protein (PV1), a caveolae-associated protein, can promote accumulation of antibodies in both the lungs and the kidneys. Here, by fine-tuning the binding affinity of an antibody toward PV1, we observe that the anti-PV1 antibody with reduced binding affinity lost the capability for kidney targeting while retaining the lung targeting activity, suggesting that binding affinity is a critical factor for kidney targeting of the anti-PV1 antibody. We next use the antibody fragment F(ab')2 targeting PV1 to assess the dual effects of rapid kidney filtration and PV1 targeting on kidney-selective targeting. Ex vivo fluorescence imaging results demonstrated that after rapidly accumulating in kidneys at 4 h, PV1-targeted F(ab')2 was continually retained in the kidney at 24 h, whereas the isotype control F(ab')2 underwent urinary elimination with significantly reduced signaling in the kidney. Confocal imaging studies confirmed the localization of PV1-targeted F(ab')2 in the kidney. In addition, the monovalent antibody fragment (Fab-C4) lost the capability for kidney homing, indicating that the binding avidity of anti-PV1 F(ab')2 is important for kidney targeting. Our findings suggest that PV1-targeted F(ab')2 might be useful as a drug carrier for renal targeting and highlight the importance of affinity optimization for tissue targeting antibodies.
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Anticorpos Monoclonais/imunologia , Cavéolas/metabolismo , Portadores de Fármacos/farmacocinética , Fragmentos Fab das Imunoglobulinas/imunologia , Rim/efeitos dos fármacos , Proteínas de Membrana/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Portadores de Fármacos/administração & dosagem , Feminino , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Rim/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors and hematological malignancies, improvements in ADC design are still desirable due to the narrow therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of many different types of biologics. PEGylation improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates. Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the mechanism of half-life extension is an important consideration for designing cytotoxic antitumor agents.
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Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaio de Imunoadsorção Enzimática , Feminino , Meia-Vida , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Polietilenoglicóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The accumulation, dissemination and clearance of monoclonal antibody-based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)-modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG-modified diabodies improved the PK of the parental diabody from a half-life of 40â¯min to over 40â¯h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody-PEG5K showing peak accumulation early on, but with the larger diabody-PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody-PEG20K-B with a hydrodynamic radius (Rh) of 6â¯nm had superior tumor uptake than the larger diabody-PEG40K-B with Rh of 12â¯nm, indicating that beyond 6â¯nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ~6â¯nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.
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Sistemas de Liberação de Medicamentos , Fragmentos de Imunoglobulinas/administração & dosagem , Neoplasias/metabolismo , Polietilenoglicóis/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Hidrodinâmica , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Camundongos , Camundongos Nus , Peso Molecular , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
RATIONALE: The parasympathetic reduction in heart rate involves the sequential activation of m2 muscarinic cholinergic receptors (m(2)Rs), pertussis toxin-sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel I(KACh). Molecular mechanisms regulating this critical signal transduction pathway are not fully understood. OBJECTIVE: To determine whether the G protein signaling regulator Rgs6/Gß5 modulates m(2)R-I(KACh) signaling and cardiac physiology. METHODS AND RESULTS: Cardiac expression of Rgs6, and its interaction with Gß5, was demonstrated by immunoblotting and immunoprecipitation. Rgs6(-/-) mice were generated by gene targeting, and the cardiac effects of Rgs6 ablation were analyzed by whole-cell recordings in isolated cardiomyocytes and ECG telemetry. Loss of Rgs6 yielded profound delays in m(2)R-I(KACh) deactivation kinetics in both neonatal atrial myocytes and adult sinoatrial nodal cells. Rgs6(-/-) mice exhibited mild resting bradycardia and altered heart rate responses to pharmacological manipulations that were consistent with enhanced m(2)R-I(KACh) signaling. CONCLUSIONS: The cardiac Rgs6/Gß5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice.
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Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Frequência Cardíaca/fisiologia , Ativação do Canal Iônico/fisiologia , Miócitos Cardíacos/fisiologia , Fibras Parassimpáticas Pós-Ganglionares/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Proteínas RGS/fisiologia , Regulação para Cima/fisiologia , Animais , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Células HEK293 , Átrios do Coração/citologia , Átrios do Coração/fisiopatologia , Frequência Cardíaca/genética , Humanos , Ativação do Canal Iônico/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Regulação para Cima/genéticaRESUMO
The type 5 G protein beta subunit (Gbeta5) can form complexes with members of the regulator of G protein signaling 7 (RGS7) family, but its relevance to neuronal G protein signaling is unclear. We found that mouse RGS7-Gbeta5 complexes bound to G protein-gated potassium channels and facilitated their functional coupling to GABA(B) receptors in neurons. Our findings identify a compartmentalization mechanism that is critical for ensuring high temporal resolution of neuronal G protein signaling.
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Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Inibição Neural/fisiologia , Neurônios/fisiologia , Proteínas RGS/metabolismo , Transmissão Sináptica/fisiologia , Animais , Baclofeno/farmacologia , Linhagem Celular , Células Cultivadas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Agonistas GABAérgicos/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/genética , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
An enhancer within intron 1 of the amyloid precursor protein gene (APPb) of zebrafish is identified functionally using a novel approach. Bacterial artificial chromosomes (BACs) were retrofitted with enhancer traps, and expressed as transgenes in zebrafish. Expression from both transient assays and stable lines were used for analysis. Although the enhancer was active in specific nonneural cells of the notochord when placed with APPb gene promoter proximal elements its function was restricted to, and absolutely required for, specific expression in neurons when juxtaposed with additional far-upstream promoter elements of the gene. We demonstrate that expression of green fluorescent protein fluorescence resembling the tissue distribution of APPb mRNA requires both the intron 1 enhancer and approximately 28 kb of DNA upstream of the gene. The results indicate that tissue-specificity of an isolated enhancer may be quite different from that in the context of its own gene. Using this enhancer and upstream sequence, polymorphic variants of APPb can now more closely recapitulate the endogenous pattern and regulation of APPb expression in animal models for Alzheimer's disease. The methodology should help functionally map multiple noncontiguous regulatory elements in BACs with or without gene-coding sequences.