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1.
Artigo em Inglês | MEDLINE | ID: mdl-32109745

RESUMO

In this study, haptens were designed to produce highly sensitive and specific monoclonal antibodies (mAb) against carbamazepine (CBZ) and its metabolite carbamazepine-10, 11-epoxide (CBZ-EP). According to the results of our competitive enzyme-linked immunosorbent assay (ic-ELISA), the half-maximum inhibitory concentration values for anti-CBZ and anti-CBZ-EP mAb were 0.18 and 0.59 ng/mL, respectively. An immunochromatographic assay (ICA) was developed for the determination of CBZ and CBZ-EP concentrations. This method can provide visible limits of detection ranging from 0.25 to 1 ng/mL, and cut-off limits ranging from 5 to 10 ng/mL, and takes 10 min to evaluate with the naked eye. Importantly, these observations were consistent with those obtained by ic-ELISA and liquid chromatography-mass spectrometry. The ICA assay represented a reliable, fast, and high-throughput method for the determination of CBZ and CBZ-EP in serum samples.


Assuntos
Anticorpos Monoclonais/química , Carbamazepina/análogos & derivados , Carbamazepina/sangue , Imunoensaio/métodos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/metabolismo , Carbamazepina/isolamento & purificação , Carbamazepina/metabolismo , Cromatografia Líquida , Humanos , Limite de Detecção , Modelos Lineares
2.
Chem Commun (Camb) ; 56(10): 1605-1607, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31939465

RESUMO

We report the first preparation of a monoclonal antibody (mAb) that can immobilize a palladium (Pd)-complex. The allylic amination reaction using a supramolecular catalyst consisting of the Pd-complex and mAb selectively gives the (R)-enantiomer product with an enantiomeric excess (ee) of 98 ± 2%. This is in sharp contrast to the reaction catalyzed by a conventional Pd-catalyst (ee < 2%).


Assuntos
Anticorpos Monoclonais/química , Complexos de Coordenação/química , Paládio/química , Compostos Alílicos/síntese química , Compostos Alílicos/química , Aminação , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Benzilaminas/síntese química , Benzilaminas/química , Catálise , Bovinos , Complexos de Coordenação/imunologia , Complexos de Coordenação/metabolismo , Reações Cruzadas/imunologia , Feminino , Gastrópodes/química , Hemocianinas/química , Camundongos Endogâmicos BALB C , Ligação Proteica , Ródio/química , Soroalbumina Bovina/química , Estereoisomerismo , Água/química
3.
Biomed Chromatogr ; 34(2): e4718, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31642080

RESUMO

There have been reports of fake artesunate (ART), which has led to deaths from untreated malaria in South East Asia. To rapidly screen for fake and adulterated ART products in the drug market, a lateral flow immunoassay (LFIA) based on a colloidal gold-monoclonal antibody probe for detection of ART within samples was developed. With this method, the calibration curve for ART was determined by the intensity ratio of the test and control bands at various ART concentrations. The linearity range was 12.5-200 µg/ml of ART. Samples were tested by the developed LFIA and can be calculated for ART contents. The levels of ART in the samples were also confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good conformance. The proposed LFIA was demonstrated to be a simple and rapid analytical method for detecting ART in the pharmaceutical formulation.


Assuntos
Anticorpos Monoclonais/metabolismo , Artesunato/análise , Imunoensaio/métodos , Artesunato/química , Artesunato/metabolismo , Artesunato/normas , Medicamentos Falsificados/análise , Medicamentos Falsificados/química , Medicamentos Falsificados/metabolismo , Desenho de Equipamento , Coloide de Ouro/química , Imunoensaio/instrumentação , Modelos Lineares
4.
Artigo em Inglês | MEDLINE | ID: mdl-31881514

RESUMO

Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) is a common method for characterizing primary sequences of monoclonal antibodies (mAbs) and their post-translational modifications (PTMs). Most methods prepare digests by incubating samples with proteases from several hours to overnight. This often induces artifacts of some modifications such as deamidation and isomerization, resulting in overestimated product-related modifications levels. Hour-long-digestion can generate complicate chromatographic profiles due to semi-cleavages or other unnecessary reactions, interfering with the quantitation of peaks of interest. On the other hand, shortening digestion-time can cause incomplete peptide cleavages, thus low sequence coverage and poor repeatability. This study applied pressure cycling technology (PCT) to tryptic digestion and PNGase F deglycosyaltion. A 0.5-h PCT assistant tryptic digestion, by alternating cycles of 10-s atmospheric pressure and 50-s high pressure (30 kpsi) at 37 °C, was evaluated and compared with two conventional digestions, 4-h and 18-h (i.e., overnight) incubations at 37 °C under atmospheric pressure. The 0.5-h PCT assistant deglycosylation was also assessed using the same conditions as those by PCT tryptic digestion. The results demonstrated the application of a 0.5-h PCT to tryptic digestion minimized modification artifacts and reduced interference with quantitation by providing a clean chromatographic profile. The 0.5-h PCT assistant deglycosylation completely removed the N-glycans from the Asn301 in the heavy chains of monoclonal antibody with no impact on the chromatographic profile of the tryptic digests.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Humanos , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121853, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31785532

RESUMO

A box-shaped or cuboid packed-bed device was used for monoclonal antibody (mAb) separation using protein A affinity chromatography. The separation efficiency of the device was compared with an equivalent column i.e. packed with same resin, and having identical bed height and bed volume. The protein A media packed cuboid device had a larger number of theoretical plates than its equivalent column, e.g. 8750/m as opposed to about 4700/m at a flow rate of 0.5 mL/min. In mAb purification experiments, the impurity flow-through and eluted mAb peaks were shaper with the cuboid device. This implied that the effective separation time and buffer consumption with this device was lower, the purified mAb pooled volume was smaller, and the mAb concentration in the pooled volume was greater. Equivalent separation efficiency could be obtained with the cuboid device using higher flow rates than that used with the column. For instance, elution peaks equivalent to those obtainable by the column could be obtained at a 5 times greater flow rate using the cuboid device. The results discussed in this paper clearly demonstrate the potential for improving the efficiency of protein A affinity chromatography based mAb purification by using a cuboid packed-bed device.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/química , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Técnicas de Cultura de Células , Cromatografia de Afinidade/instrumentação , Cricetinae , Cricetulus , Desenho de Equipamento
6.
PLoS One ; 14(12): e0226553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877149

RESUMO

BACKGROUND: Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer. METHODOLOGY: ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint). RESULTS: ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues. CONCLUSIONS/SIGNIFICANCE: Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Peptidil Dipeptidase A/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Idoso , Anticorpos Monoclonais/metabolismo , Epitopos/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/química , Fenótipo
7.
Life Sci ; 239: 117052, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31733318

RESUMO

AIMS: A combination of biomarker and instrument technology diagnosis methods, especially antigen-targeted imaging methods, is required to increase the accuracy of the diagnosis of cancer. Currently, the targeting efficiency is limited by the conjugation methods used for the conjugation of antibodies and imaging materials. Here, a simple strategy for the conjugation of a probe and a single-chain fragment antibody (scFv) that does not change the characteristics of the antibody was shown. MAIN METHODS: An ScFv was conjugated with superparamagnetic iron oxide (SPIO) or indocyanine green (ICG) via a linker by utilizing the reaction between cysteine and maleimide. The characterization of the probe was performed by flow cytometry, confocal imaging, optical imaging and magnetic resonance imaging (MRI). KEY FINDINGS: After conjugation, the scFv retained high affinity, antigen specificity, and strong internalization ability. The application of the conjugated probe was also confirmed by optical imaging and MRI. SIGNIFICANCE: The proposed strategy provides a simple method for the production of high efficiency antigen-targeted imaging probes for tumor diagnosis.


Assuntos
Anticorpos Monoclonais/química , Imagem por Ressonância Magnética/métodos , Anticorpos de Cadeia Única/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Meios de Contraste , Compostos Férricos/química , Citometria de Fluxo , Humanos , Verde de Indocianina/química , Nanopartículas de Magnetita , Anticorpos de Cadeia Única/metabolismo
8.
Immunity ; 51(4): 724-734.e4, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586542

RESUMO

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Infecções por HIV/imunologia , HIV/fisiologia , Polissacarídeos/química , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Antígenos CD4/metabolismo , Células Cultivadas , Cristalização , Cristalografia por Raios X , Modelos Animais de Doenças , Glicosilação , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Macaca mulatta , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
9.
Aquat Toxicol ; 216: 105321, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31586886

RESUMO

Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.


Assuntos
Organismos Aquáticos/metabolismo , Biomarcadores/análise , Estrogênios/toxicidade , Linguado/metabolismo , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Compostos Benzidrílicos/toxicidade , Exposição Ambiental , Estradiol/análise , Etinilestradiol/toxicidade , Linguado/sangue , Fenóis/toxicidade , Vitelogeninas/sangue
10.
Adv Exp Med Biol ; 1172: 97-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628653

RESUMO

The IL-17 family in humans consists of six distinct cytokines (IL-17A-F) that can interact with five IL-17 receptors (IL-17RA-E). The interaction between these cytokines and their receptors are critical in mediating host defenses while also making major contributions to inflammatory and autoimmune responses as demonstrated through both in vitro and in vivo experiments as well as human clinical trials. Inhibition of the IL-17A/IL-17RA interaction by monoclonal antibodies has also displayed remarkable efficacies in clinical trials against psoriasis and other autoimmune diseases. Recently, we and others reported the identification and characterization of both small-molecule and peptide IL-17A antagonists. These non-antibody IL-17A antagonists can effectively and selectively disrupt the IL-17A/IL-17RA complex and may provide alternative modalities to treat IL-17-related autoimmune and inflammatory diseases. This chapter summarizes the reported crystal structures of the IL-17 cytokines, their complexes with IL-17RA, and their complexes with both monoclonal antibodies as well as small-molecule and peptide antagonists.


Assuntos
Interleucina-17 , Receptores de Interleucina-17 , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Doenças Autoimunes/imunologia , Cristalização , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Interleucina-17/imunologia , Receptores de Interleucina-17/antagonistas & inibidores , Receptores de Interleucina-17/química , Receptores de Interleucina-17/imunologia
11.
Int J Nanomedicine ; 14: 8013-8031, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632015

RESUMO

Introduction: This study was conducted to evaluate OX26-PEG-coated gold nanoparticles (GNPs) (OX26@GNPs) as a novel targeted nanoparticulate system on cell survival after ischemic stroke. Materials and methods: Dynamic light scattering (DLS), zeta sizer, and transmission electron microscopy (TEM) were performed to characterize the OX26@GNPs. The effect of OX26@GNPs on infarct volume, neuronal loss, and necroptosis was evaluated 24 h after reperfusion using 2, 3,5-Triphenyltetrazolium chloride (TTC) staining, Nissl staining and Western blot assay, respectively. Results: Conjugation of OX26-PEG to the surface of the 25 nm colloidal gold particles increased their size to 32±2 nm, while a zeta potential change of -40.4 to 3.40 mV remarkably increased the stability of the nanoparticles. Most importantly, OX26@GNPs significantly increased the infarcted brain tissue, while bare GNPs and PEGylated GNPs had no effect on the infarct volume. However, our results indicated an extension of necroptotic cell death, followed by cell membrane damage. Conclusion: Collectively, our results showed that the presently formulated OX26@GNPs are not suitable nanocarriers nor contrast agents under oxidative stress for the diagnosis and treatment of ischemic stroke. Moreover, our findings suggest that the cytotoxicity of GNPs in the brain is significantly associated with their surface charge.


Assuntos
Isquemia Encefálica/líquido cefalorraquidiano , Portadores de Fármacos/química , Coloide de Ouro/química , Acidente Vascular Cerebral/diagnóstico , Animais , Anticorpos Monoclonais/metabolismo , Isquemia Encefálica/complicações , Coloide de Ouro/administração & dosagem , Coloide de Ouro/toxicidade , Humanos , Hidrodinâmica , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Ratos Wistar , Eletricidade Estática , Acidente Vascular Cerebral/complicações
13.
Monoclon Antib Immunodiagn Immunother ; 38(5): 209-212, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31603743

RESUMO

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.


Assuntos
Enterotoxinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Western Blotting , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática/instrumentação , Feminino , Hibridomas , Camundongos Endogâmicos BALB C
14.
Anal Chim Acta ; 1089: 1-18, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627805

RESUMO

Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products.


Assuntos
Anticorpos Monoclonais/análise , Medicamentos Biossimilares/análise , Glicoproteínas/análise , Polissacarídeos/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Medicamentos Biossimilares/química , Medicamentos Biossimilares/metabolismo , Cromatografia Líquida , Glicoproteínas/química , Glicosilação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Infliximab/análise , Infliximab/química , Infliximab/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
15.
Monoclon Antib Immunodiagn Immunother ; 38(5): 230-233, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31535919

RESUMO

Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Ursidae , Animais , Anticorpos Monoclonais/metabolismo , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos/métodos , Epitopos/metabolismo , Imuno-Histoquímica , Rim/imunologia , Glicoproteínas de Membrana/genética , Mutação Puntual
16.
Nat Commun ; 10(1): 4186, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519915

RESUMO

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.


Assuntos
Inflamação/metabolismo , Melanoma/metabolismo , Anticorpos Monoclonais/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Humanos , Imunoterapia , Técnicas In Vitro , Inflamação/imunologia , Melanoma/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/metabolismo
18.
Anal Chim Acta ; 1079: 252-259, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387718

RESUMO

The concept of coupling of size-exclusion HPLC with ICP/MS (SEC-ICP/MS) was first applied in this work as a novel approach in the biotechnology field to assess metal binding to Immunoglobulin G (IgG) mAbs. This method can be used to probe the mechanism and biophysical properties of metal-protein interactions to gain a deeper understanding of the potential impact of metals during drug product manufacturing. Two IgG1s and one IgG2 drugs were investigated. Cu2+ was selected as the metal of interest due to its known ability to form strong complexes with organic molecules and to bind and enhance the degradation of mAbs. Instrument and separation conditions (interface, columns, and mobile phase) were studied for the separation of the protein-metal bound and unbound fractions of a bovine superoxide dismutase (SOD) standard prior to on-line detection of the mAb-metal (Cu) binding. The SEC-ICP/MS method was used to show copper binding by biotherapeutics by comparing the retention times of the protein by SEC and the metal by ICP/MS, to see if they co-elute at the same time. The approach developed offers considerable advantages over methods based on ultrafiltration followed by off-line metal determination in terms of speed, simplicity, precision and selectivity regarding the molecular weight of the complexes involved. In conjunction with other techniques, this method may provide in-depth knowledge of metal-induced mAb degradation mechanisms in biologics process development, be used as an analytical tool for mAb manufacturing in the cell culture process, and be applied during various stages of drug product manufacturing to gain a deeper understanding of the potential impact of metals during biotherapeutic development.


Assuntos
Anticorpos Monoclonais/metabolismo , Cobre/metabolismo , Imunoglobulina G/metabolismo , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Ligação Proteica
19.
J Biotechnol ; 305: 51-60, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31442501

RESUMO

Monoclonal antibody (mAb) fragmentation is a well-known degradation pathway that results in product loss and can significantly impact product quality, efficacy, or even cause immunogenic reactions, thus potentially endangering patients' health. It is recognised that residual proteases present among host cell proteins (HCPs) such as those expressed by Chinese Hamster Ovary (CHO) can induce fragmentation, and failure of their complete removal during downstream processing could cause fragmentation during mAb production and in the final drug product. We identified, using a protease inhibitor screen, an aspartic protease that contributes to proteolytic fragmentation of partially purified mAbs in multiple projects. Subsequent LC-MS analysis indicated that cathepsin D, a typical aspartic protease, was responsible for the observed fragmentation of in-process samples. To address the issue, an alternative chromatography wash was implemented at the capture step and has been demonstrated to be an effective and scalable solution to mitigate the residual cathepsin D associated fragmentation risk. Furthermore, a near real time targeted mass spectrometry method has been developed to proactively monitor the presence of cathepsin D during upstream and downstream process. Our approach demonstrated an emerging HCP mitigation strategy through integrated upstream and downstream involvement and holds great promise for a range of future applications.


Assuntos
Catepsina D/metabolismo , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Estabilidade de Medicamentos , Espectrometria de Massas , Proteólise
20.
ACS Appl Mater Interfaces ; 11(36): 33380-33389, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31433617

RESUMO

Peptides isolated from phage display libraries are powerful reagents for small-molecule immunoassay; however, their application as phage-borne peptides is significantly limited by the biological nature of the phage. Here, we present the use of lysine scaffold to prepare a series of different valence peptides to serve as replacements for phage-borne peptides. Benzothiostrobin was selected as a model analyte, the cyclic benzothiostrobin-peptidomimetic in the form of monomer, dendrimer-like dimer, and tetramer were designed and synthesized. Compared with the monomer, the affinity of dendrimer-like dimer and tetramer increased 1.87 and 13.6 times, respectively, as determined by isothermal titration calorimetry (ITC). A novel inner filter effect immunoassay (IFE-IA) with positive readout was developed for benzothiostrobin detection utilizing the peptidomimetics attached to upconversion nanoparticles (UCNPs) as energy donor and monoclonal antibody (mAb)-labeled urchin-like gold nanoflowers (AuNFs) as energy absorber, respectively. The sensitivity of the assay based on dendrimer-like tetramer was approximately 6 and 3 times higher than monomer and dendrimer-like dimer, respectively. After optimization, 50% saturation of the signal (SC50) and detection range (SC10 to SC90) of the IFE-IA based on dendrimer-like tetramer were 11.81 ng mL-1 and 2.04-106.17 ng mL-1, respectively. The IFE-IA also shows good accuracy for the detection of benzothiostrobin in authentic samples.


Assuntos
Dendrímeros/química , Nanopartículas Metálicas/química , Peptídeos/química , Acrilatos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Benzotiazóis/química , Ouro/química , Imunoensaio , Nanopartículas Metálicas/ultraestrutura , Peptidomiméticos , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
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