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1.
J Chromatogr A ; 1604: 460474, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31493850

RESUMO

Biomimetic affinity chromatography with short peptide ligands is a developing technology, which has great potential for antibody separation and purification. In this study, a tetrapeptide library with critical residues of natural ligands to hIgG was constructed and a novel tetrapeptide ligand (Ac-FYHE) with high LibDock scores was selected by molecular docking. Then, Ac-FYHE ligand was linked to agarose bead to prepare a new chromatography resin. The properties of antibody adsorption were measured and evaluated by static/dynamic adsorption. It was found that the resin with ligand Ac-FYHE has high binding capacity and selectivity for hIgG. The results showed the Qm-hIgG of Ac-FYHE-4FF resin was 87.9 mg/g resin while the Qm-BSA of this resin was only 16.5 mg/g resin at pH 7.0. Moreover, at pH 7.0, Q10% of Ac-FYHE-4FF resin was 24.1 mg/mL for hIgG but just 2.1 mg/mL for BSA, which presented high selectivity of the screened resin at pH 7.0. Subsequently, the adsorption and separation properties of the Ac-FYHE-4FF resin were further investigated. As a result, with the addition of 0.5 M NaCl, Qm decreased by less than 20% but Qm decreased by 70% with the addition of 50% (v/v) ethylene glycol, which indicated that hydrophobic interaction would be the driving force for the binding between resin and hIgG. Besides, pH 7.5 and pH 4.5 could be the optimal loading and elution condition for hIgG, respectively. Finally, the Ac-FYHE-4FF resin was applied to separate mAb or/and hIgG from BSA containing feedstock, CHO cell culture supernatant and human serum, and the purity and recovery were both more than 90% with only one-step separation. The results indicate that the Ac-FYHE-4FF resin developed in this work would be promising for antibody separation and purification.


Assuntos
Anticorpos/isolamento & purificação , Biomimética , Técnicas de Química Analítica/métodos , Cromatografia de Afinidade , Adsorção , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento Molecular , Peptídeos/química , Sefarose/química
2.
Cancer Sci ; 110(9): 2722-2733, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31461572

RESUMO

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.


Assuntos
Proteínas Ligadas por GPI/imunologia , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetulus , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/isolamento & purificação , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular/métodos , Neoplasias/patologia , Biblioteca de Peptídeos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Compostos Radiofarmacêuticos/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio
3.
Chem Commun (Camb) ; 55(61): 8975-8978, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290492
4.
Adv Exp Med Biol ; 1140: 225-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347050

RESUMO

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.


Assuntos
Anticorpos Monoclonais/química , Sequenciamento de Nucleotídeos em Larga Escala , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Cricetinae , Cricetulus , Espectrometria de Massas
5.
Cancer Sci ; 110(10): 3340-3349, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342590

RESUMO

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion-metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET-knockout cells were prepared and reconstituted with WT-MET or V370D-MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT-MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D-MET cells. The V370D mutation abrogated HGF-dependent drug resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). Compared with WT-MET cells, V370D-MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth-stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D-MET and HGF was reduced compared with that for WT-MET. Further analysis of the association between V370D-MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D-MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss-of-function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Resistencia a Medicamentos Antineoplásicos , Técnicas de Inativação de Genes , Humanos , Mutação com Perda de Função , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas c-met/metabolismo , Ativação Transcricional
6.
Eur J Med Chem ; 180: 1-14, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31288149

RESUMO

SAR studies on bicalutamide, enobosarm and enzalutamide analogues, functionalised with polyfluorinated groups, is presented. Among the novel bicalutamide and enobosarm derivatives synthesised, several displayed significantly improved in vitro anticancer activity, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145 and 22Rv1), showing up to 48-fold increase in comparison with the parent structures. In particular, SF5 enobosarm analogues were found to be most potent compounds, full AR antagonists and with favourable ADME properties. The most promising compound (48a) was evaluated for its in vivo efficacy in PC xenograft mouse model (22Rv1) with results comparable to the standard-of-care docetaxel.


Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Nitrilos/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Compostos de Tosil/farmacologia , Anilidas/síntese química , Anilidas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/farmacologia , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Nitrilos/síntese química , Nitrilos/química , Feniltioidantoína/síntese química , Feniltioidantoína/química , Feniltioidantoína/farmacologia , Neoplasias da Próstata/patologia , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Compostos de Tosil/síntese química , Compostos de Tosil/química
7.
Pharm Res ; 36(9): 137, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332533

RESUMO

PURPOSE: Pitt Hopkins Syndrome (PTHS) is a rare genetic disorder caused by mutations of a specific gene, transcription factor 4 (TCF4), located on chromosome 18. PTHS results in individuals that have moderate to severe intellectual disability, with most exhibiting psychomotor delay. PTHS also exhibits features of autistic spectrum disorders, which are characterized by the impaired ability to communicate and socialize. PTHS is comorbid with a higher prevalence of epileptic seizures which can be present from birth or which commonly develop in childhood. Attenuated or absent TCF4 expression results in increased translation of peripheral ion channels Kv7.1 and Nav1.8 which triggers an increase in after-hyperpolarization and altered firing properties. METHODS: We now describe a high throughput screen (HTS) of 1280 approved drugs and machine learning models developed from this data. The ion channels were expressed in either CHO (KV7.1) or HEK293 (Nav1.8) cells and the HTS used either 86Rb+ efflux (KV7.1) or a FLIPR assay (Nav1.8). RESULTS: The HTS delivered 55 inhibitors of Kv7.1 (4.2% hit rate) and 93 inhibitors of Nav1.8 (7.2% hit rate) at a screening concentration of 10 µM. These datasets also enabled us to generate and validate Bayesian machine learning models for these ion channels. We also describe a structure activity relationship for several dihydropyridine compounds as inhibitors of Nav1.8. CONCLUSIONS: This work could lead to the potential repurposing of nicardipine or other dihydropyridine calcium channel antagonists as potential treatments for PTHS acting via Nav1.8, as there are currently no approved treatments for this rare disorder.


Assuntos
Di-Hidropiridinas/farmacologia , Reposicionamento de Medicamentos/métodos , Hiperventilação/tratamento farmacológico , Deficiência Intelectual/tratamento farmacológico , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Teorema de Bayes , Células CHO , Cricetulus , Di-Hidropiridinas/química , Facies , Células HEK293 , Humanos , Canal de Potássio KCNQ1/metabolismo , Aprendizado de Máquina , Bloqueadores dos Canais de Potássio/química , Bibliotecas de Moléculas Pequenas/química , Bloqueadores dos Canais de Sódio/química , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
8.
BMC Complement Altern Med ; 19(1): 153, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262287

RESUMO

BACKGROUND: Rhus trilobata Nutt. (Anacardiaceae) (RHTR) is a plant of Mexico that is traditionally used as an alternative treatment for several types of cancer. However, the phytochemical composition and potential toxicity of this plant have not been evaluated to support its therapeutic use. Therefore, this study aimed to evaluate the biological activity of RHTR against colorectal adenocarcinoma cells, determine its possible acute toxicity, and analyze its phytochemical composition. METHODS: The traditional preparation was performed by decoction of stems in distilled water (aqueous extract, AE), and flavonoids were concentrated with C18-cartridges and ethyl acetate (flavonoid fraction, FF). The biological activity was evaluated by MTT viability curves and the TUNEL assay in colorectal adenocarcinoma (CACO-2), ovarian epithelium (CHO-K1) and lung/bronchus epithelium (BEAS-2B) cells. The toxicological effect was determined in female BALB/c mice after 24 h and 14 days of intraperitoneal administration of 200 mg/kg AE and FF, respectively. Later, the animals were sacrificed for histopathological observation of organs and sera obtained by retro-orbital bleeding for biochemical marker analysis. Finally, the phytochemical characterization of AE and FF was conducted by UPLC-MSE. RESULTS: In the MTT assays, AE and FF at 5 and 18 µg/mL decreased the viability of CACO-2 cells compared with cells treated with vehicle or normal cells (p ≤ 0.05, ANOVA), with changes in cell morphology and the induction of apoptosis. Anatomical and histological analysis of organs did not reveal important pathological lesions at the time of assessment. Additionally, biochemical markers remained normal and showed no differences from those of the control group after 24 h and 14 days of treatment (p ≤ 0.05, ANOVA). Finally, UPLC-MSE analysis revealed 173 compounds in AE-RHTR, primarily flavonoids, fatty acids and phenolic acids. The most abundant compounds in AE and FF were quercetin and myricetin derivates (glycosides), methyl gallate, epigallocatechin-3-cinnamate, ß-PGG, fisetin and margaric acid, which might be related to the anticancer properties of RHTR. CONCLUSION: RHTR exhibits biological activity against cancer cells and does not present adverse toxicological effects during its in vivo administration, supporting its traditional use.


Assuntos
Antineoplásicos Fitogênicos/análise , Rhus/química , Animais , Antioxidantes/análise , Células CHO , Células CACO-2 , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Flavonoides/análise , Humanos , Medicina Tradicional , México , Camundongos Endogâmicos BALB C , Fitoterapia , Extratos Vegetais/análise , Extratos Vegetais/uso terapêutico , Extratos Vegetais/toxicidade , Polifenóis/análise , Rhus/toxicidade
9.
AAPS PharmSciTech ; 20(6): 246, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286304

RESUMO

Scale-down models are indispensable and crucial tools for process understanding and continuous process improvement in product life-cycle management. In this study, a scale-down model representing commercial-scale cell culture process of adalimumab biosimilar HS016 was first developed based on constant power per volume (P/V) principle and then qualified by multivariate data analysis (MVDA) and equivalence test method. The trajectories of the bench-scale process lie in the middle of the control range of large-scale process, built by multivariate evolution model based on nutrients, metabolites, and process performance datasets. This indicates that the small-scale process performance is comparable with that of the full-scale process. The final product titer, integrated viable cell density (iVCD), viability, aggregates, acid peak content, total afucosylation level, and high mannose content recognized as key process attributes (KPAs) or critical quality attributes (CQAs) were equivalent across the scales upon comparison using equivalence test method. The qualified scale-down model was then used for process characterization using a definitive screening design (DSD) where five independent variables including pH, shifted temperature, inoculation seeding density, viable cell density (VCD) at first feeding, VCD at temperature shift were evaluated. Three quadratic polynomial models for final product titer, aggregation, and high mannose were then established using the DSD results. The design space was finally developed using a probability-based Monte Carlo simulation method and was verified with the operation setpoint and worst-case condition. The case study presented in this report shows a feasible roadmap for cell culture process characterization.


Assuntos
Desenvolvimento de Medicamentos , Adalimumab/química , Animais , Células CHO , Química Farmacêutica , Cricetinae , Cricetulus , Modelos Estatísticos , Método de Monte Carlo , Análise Multivariada , Temperatura Ambiente
10.
Eur J Med Chem ; 178: 243-258, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185414

RESUMO

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against Aß1-42 oligomers, was a potent and selective CB2 ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB2 inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB2 receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system.


Assuntos
Benzofuranos/farmacologia , Inibidores da Colinesterase/farmacologia , Fatores Imunológicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Benzofuranos/síntese química , Benzofuranos/química , Benzofuranos/metabolismo , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Células CHO , Domínio Catalítico , Linhagem Celular Tumoral , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Cricetulus , Desenho de Drogas , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Ligação Proteica , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
11.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1071-1078, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232003

RESUMO

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human ß-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.


Assuntos
Íntrons , Animais , Animais Geneticamente Modificados , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Transfecção
12.
J Chromatogr A ; 1602: 284-299, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31230875

RESUMO

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48-57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4-7.8 10-7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.


Assuntos
Anticorpos/metabolismo , Afinidade de Anticorpos , Peptídeos/química , Peptoides/química , Adsorção , Álcalis/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteólise , Temperatura Ambiente
13.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 554-563, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205018

RESUMO

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.


Assuntos
Anticorpos Monoclonais Humanizados/química , Complexo Antígeno-Anticorpo/química , Antineoplásicos Imunológicos/química , Neoplasias/terapia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cristalização , Cristalografia por Raios X/métodos , Feminino , Humanos , Domínios Proteicos , Receptor ErbB-2/imunologia
14.
Dokl Biochem Biophys ; 485(1): 101-103, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201624

RESUMO

In this paper, we present an approach to optimize the heterologous expression of the receptor tyrosine kinase IRR, which further simplifies the purification of the IRR from the medium and increases the final yield. The approach proposed by us can find application in the biotechnological production of other large-scale recombinant proteins produced for medical purposes.


Assuntos
Receptor de Insulina/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Domínios Proteicos , Receptor de Insulina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
15.
BMC Infect Dis ; 19(1): 482, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146699

RESUMO

BACKGROUND: To assess the immune persistence conferred by a Chinese hamster ovary (CHO)-derived hepatitis B vaccine (HepB) 17 to 20 years after primary immunization during early life. METHODS: Participants born between 1997 and 1999 who received a full course of primary vaccination with HepB (CHO) and who had no experience with booster vaccination were enrolled. Blood samples were required from each participant for measurement of hepatitis B surface antibody (anti-HBs), surface antigen and core antibody levels. For those who possessed an anti-HBs antibody < 10 mIU/mL, a single dose of HepB was administered, and 30 days later, serum specimens were collected to assess the booster effects. RESULTS: A total of 1352 participants were included in this study. Of these, 1007 (74.5%) participants could retain an anti-HBs antibody ≥10 mIU/mL, with a geometric mean concentration (GMC) of 57.4 mIU/mL. HBsAg was detected in six participants, resulting in a HBsAg carrier rate of 0.4% (6/1352). Of those participants with anti-HBs antibodies < 10 mIU/mL, after a challenge dose, 231 (93.1%) presented an anti-HBs antibody ≥10 mIU/mL, with a GMC of 368.7 mIU/mL. A significant increase in the anti-HBs positive rate (≥ 10 mIU/mL) after challenge was observed in participants with anti-HBs antibodies between 2.5 and 10 mIU/mL and participants boosted with HepB (CHO), rather than those with anti-HBs antibodies < 2.5 mIU/mL and those boosted with HepB (SC). CONCLUSION: Since satisfactory immune protection against HBV infection conferred by primary vaccination administered 17-20 years ago was demonstrated, there is currently no urgent need for booster immunization.


Assuntos
Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Imunização Secundária , Prevenção Primária , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Adolescente , Adulto , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Seguimentos , Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Recém-Nascido , Masculino , Prevenção Primária/métodos , Estudos Retrospectivos , Fatores de Tempo , Adulto Jovem
16.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
17.
Eur J Med Chem ; 179: 78-83, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31238252

RESUMO

Rising resistance to conventional therapies for malaria has led to the search for novel drugs and strategies with distinct mechanisms of action that may overcome this. Ferroquine is currently the gold standard as far as antimalarial metal-based drugs are concerned and is currently in phase IIb clinical trials as part of the MMV pipeline (in partnership with Sanofi) of antimalarials. It is assumed to inhibit haemozoin formation like chloroquine and maintain its activity in the resistant strain. Here we report two ferroquine-derived polyamines that target a resistant strain of Plasmodium falciparum. Furthermore, upon treatment of Plasmodium falciparum with a ferroquine-derived polyamine, cellular haem fractionation experiments reveal that the inhibition of haemozoin formation is likely not the mechanism responsible for their activity, an important result to aid further clinical development.


Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Poliaminas/farmacologia , Aminoquinolinas/química , Animais , Antimaláricos/síntese química , Antimaláricos/química , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Compostos Ferrosos/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Poliaminas/síntese química , Poliaminas/química , Relação Estrutura-Atividade
18.
Chem Biol Interact ; 309: 108712, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31201777

RESUMO

The recent intentional use of nerve agents and pesticides in Europe and Afghanistan highlights the need for an effective countermeasure against organophosphates (OP) toxins. The most developed pretreatment candidate to date is plasma (native) human butyrylcholinesterase (HuBChE), which is limited in availability and because of its 1:1 stoichiometry with OPs, a large dose will present challenges when delivered parenterally both in terms of pharmacokinetics and manageability in the field. A tetrameric recombinant (r) form of human BChE produced in CHO-K1 cells with similar structure, in vivo stability and antidotal efficacy as the native form, has been developed to deliver rHuBChE as an aerosol (aer) to form a pulmonary bioshield capable of neutralizing inhaled OPs in situ and prevent AChE inhibition in the blood and in the brain; the latter associated with the symptoms of OP toxicity. Previous proof-of-concept macaque studies demonstrated that delivery of 9 mg/kg using a microsprayer inserted down the trachea, resulted in protection against an inhaled dose of 15ug/kg of aer-paraoxon (aer-Px) given 72 h later. In the present studies, pulmonary delivery of rHuBChE in macaques was achieved using Aerogen vibrating mesh nebulizers, similar to that used for human self-administration. The promising findings indicate that despite the poor lung deposition observed in macaques using nebulizers (13-20%), protective levels of RBC-AChE were still present in the blood even when exposure aer-Px (55 µg/kg) was delayed for five days. This long term retention of 5 mg/kg rHuBChE deposited in the lung bodes well for the use of an aer-rHuBChE pretreatment in humans where a user-friendly customized nebulizer with increased lung deposition up to 50% will provide even longer protection at a lower dose.


Assuntos
Aerossóis/química , Butirilcolinesterase/química , Paraoxon/química , Animais , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Pulmão/metabolismo , Macaca , Masculino , Nebulizadores e Vaporizadores , Paraoxon/toxicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química
19.
Nat Commun ; 10(1): 2766, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235692

RESUMO

A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Modelos Genéticos , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Células CHO , Simulação por Computador , Cricetulus , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retroalimentação Fisiológica , Regulação da Expressão Gênica/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Puromicina/farmacologia , Puromicina/uso terapêutico , Processos Estocásticos
20.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035668

RESUMO

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.


Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ouabaína/farmacologia , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Expressão Gênica , Ligação Proteica , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/metabolismo
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