Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.123
Filtrar
1.
Cell Host Microbe ; 28(4): 516-525.e5, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32941787

RESUMO

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Formação de Anticorpos , Betacoronavirus/genética , Feminino , Células HEK293 , Humanos , Imunogenética , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Pandemias , Análise de Sequência , Glicoproteína da Espícula de Coronavírus/imunologia
2.
PLoS One ; 15(6): e0234508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555732

RESUMO

Dengue virus (DENV) infection remains a major public health concern in many parts of the world, including Southeast Asia and the Americas. Sri Lanka experienced its largest dengue outbreak in 2017. Neurological symptoms associated with DENV infection have increasingly been reported in both children and adults. Here, we characterize DENV type 2 (DENV-2) strains, which were isolated from cerebrospinal fluid (CSF) and/or serum of patients with dengue encephalitis. Acute serum and CSF samples from each patient were subjected to dengue-specific non-structural protein 1 (NS1) antigen test, IgM and IgG enzyme-linked immunosorbent assay (ELISA), virus isolation, conventional and real-time polymerase chain reaction (PCR), and next-generation sequencing (NGS). Among the 5 dengue encephalitis patients examined, 4 recovered and 1 died. DENV-2 strains were isolated from serum and/or CSF samples of 3 patients. The highest viral genome levels were detected in the CSF and serum of the patient who succumbed to the illness. A phylogenetic tree revealed that the DENV-2 isolates belonged to a new clade of cosmopolitan genotype and were genetically close to strains identified in China, South Korea, Singapore, Malaysia, Thailand, and the Philippines. According to the NGS analysis, greater frequencies of nonsynonymous and synonymous mutations per gene were identified in the nonstructural genes. The full genomes of serum- and CSF-derived DENV-2 from the same patient shared 99.7% similarity, indicating that the virus spread across the blood-brain barrier. This is the first report to describe neurotropic DENV-2 using whole-genome analysis and to provide the clinical, immunological, and virological characteristics of dengue encephalitis patients during a severe dengue outbreak in Sri Lanka in 2017.


Assuntos
Dengue/genética , Encefalite/genética , Genoma Viral/genética , Proteínas não Estruturais Virais/genética , Adulto , Criança , Dengue/líquido cefalorraquidiano , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Encefalite/líquido cefalorraquidiano , Encefalite/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/genética , Imunoglobulina M/sangue , Imunoglobulina M/genética , Masculino , Adulto Jovem
3.
PLoS One ; 15(5): e0232713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379792

RESUMO

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Becaplermina/imunologia , Desenho Assistido por Computador , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Modelos Moleculares , Mutação , Conformação Proteica , Propriedades de Superfície , Viscosidade
4.
Nat Biotechnol ; 38(6): 715-721, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32231335

RESUMO

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.


Assuntos
Anticorpos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , DNA/análise , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoglobulina G/genética , Camundongos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
5.
PLoS One ; 15(4): e0230818, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315311

RESUMO

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.


Assuntos
Força Muscular/genética , Músculo Esquelético/fisiologia , Atrofia Muscular/genética , Miostatina/genética , Actinas/genética , Animais , Extremidades/fisiologia , Fêmur/fisiologia , Expressão Gênica/genética , Guanidinoacetato N-Metiltransferase/genética , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Força Muscular/fisiologia , Atrofia Muscular/fisiopatologia , Voo Espacial/métodos , Ausência de Peso
6.
J Biotechnol ; 312: 11-22, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32114154

RESUMO

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.


Assuntos
Células CHO , Engenharia Celular/métodos , Engenharia Genética/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO/metabolismo , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Via Secretória/genética , Via Secretória/fisiologia
7.
Proc Natl Acad Sci U S A ; 117(14): 7981-7989, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209664

RESUMO

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Feto/imunologia , Feto/virologia , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Engenharia de Proteínas , RNA Viral/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
8.
Cell ; 180(5): 895-914.e27, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142680

RESUMO

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias/tratamento farmacológico , Proclorperazina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apresentação do Antígeno/efeitos dos fármacos , Biópsia , Cetuximab/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Xenoenxertos , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Trastuzumab/farmacologia
9.
PLoS Negl Trop Dis ; 14(3): e0007803, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203503

RESUMO

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.


Assuntos
Anticorpos Monoclonais/farmacologia , Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Salmonella/efeitos dos fármacos , Administração Oral , África , Animais , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Hibridomas , Imunização Passiva , Imunoglobulina A Secretora , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados , Salmonella typhimurium/efeitos dos fármacos
10.
PLoS One ; 15(3): e0229027, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182240

RESUMO

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability.


Assuntos
Substituição de Aminoácidos , Imunoglobulina G/química , Anticorpos Monoclonais/química , Varredura Diferencial de Calorimetria , Linhagem Celular , Desenho de Fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/genética , Agregados Proteicos/efeitos dos fármacos , Domínios Proteicos , Estabilidade Proteica
11.
PLoS Pathog ; 16(2): e1008083, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092122

RESUMO

Antibody functions such as neutralization require recognition of antigen by the Fab region, while effector functions are additionally mediated by interactions of the Fc region with soluble factors and cellular receptors. The efficacy of individual antibodies varies based on Fab domain characteristics, such as affinity for antigen and epitope-specificity, and on Fc domain characteristics that include isotype, subclass, and glycosylation profile. Here, a series of HIV-specific antibody subclass and hinge variants were constructed and tested to define those properties associated with differential effector function. In the context of the broadly neutralizing CD4 binding site-specific antibody VRC01 and the variable loop (V3) binding antibody 447-52D, hinge truncation and extension had a considerable impact on the magnitude of phagocytic activity of both IgG1 and IgG3 subclasses. The improvement in phagocytic potency of antibodies with extended hinges could not be attributed to changes in either intrinsic antigen or antibody receptor affinity. This effect was specific to phagocytosis and was generalizable to different phagocytes, at different effector cell to target ratios, for target particles of different size and composition, and occurred across a range of antibody concentrations. Antibody dependent cellular cytotoxicity and neutralization were generally independent of hinge length, and complement deposition displayed variable local optima. In vivo stability testing showed that IgG molecules with altered hinges can exhibit similar biodistribution and pharmacokinetic profiles as IgG1. Overall, these results suggest that when high phagocytic activity is desirable, therapeutic antibodies may benefit from being formatted as human IgG3 or engineered IgG1 forms with elongated hinges.


Assuntos
Anticorpos Neutralizantes , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Anti-HIV , HIV-1/imunologia , Éxons Codificadores da Região de Dobradiça , Fagocitose/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Células HEK293 , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia
12.
Mol Biotechnol ; 62(4): 240-251, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32108286

RESUMO

In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.


Assuntos
Imunoglobulina G/biossíntese , Agricultura Molecular/métodos , Mucuna/genética , Agrobacterium tumefaciens/genética , Expressão Gênica , Técnicas de Transferência de Genes/instrumentação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Mucuna/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Toxoplasma/imunologia , Transformação Bacteriana
13.
J Chromatogr A ; 1617: 460836, 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31973931

RESUMO

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.


Assuntos
Anticorpos Biespecíficos/química , Cromatografia , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/genética , Proteínas Recombinantes de Fusão/química , Anticorpos de Cadeia Única/genética
14.
J Biosci Bioeng ; 129(1): 121-128, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31303495

RESUMO

Chromosomes in Chinese hamster ovary (CHO) cells are labile. We have shown that high-chromosome-number CHO cells have greater potential to become robust producers of recombinant proteins. One explanation being the increase in transgene integration sites. However, high-chromosome-number cell clones produce more IgG3 following culture of single-cell clones, even under conditions that yield the same number of integrations as cells with normal chromosome numbers. Here, we characterized high-chromosome-number cells by transcriptome analysis. RNA standards were used to normalize transcriptomes of cells that had different chromosome numbers. Our results demonstrate that the mRNA ratio of ß-actin and many other genes in high-chromosome-number cells to that in normal-chromosome-number cells per cell (normalized to RNA standards) was smaller than the equivalent genomic size and cell volume ratios. Many genes encoding membrane proteins are more highly expressed in high-chromosome-number cells, probably due to differences in cell size caused by the increase in chromosomes. In addition, genes related to histone modification and lipid metabolism are differentially expressed. The reduced transcript level required per protein produced in total and the different intracellular signal transductions might be key factors for antibody production.


Assuntos
Células CHO/metabolismo , Cromossomos/genética , Imunoglobulina G/biossíntese , RNA Mensageiro/genética , Animais , Células CHO/citologia , Cromossomos/metabolismo , Cricetinae , Cricetulus , Expressão Gênica , Imunoglobulina G/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transcriptoma
15.
Nucleic Acids Res ; 48(2): e8, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31752022

RESUMO

Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.


Assuntos
Anticorpos Anti-Idiotípicos/isolamento & purificação , Desmogleína 3/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/isolamento & purificação , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Química Click , Cicloparafinas/química , Desmogleína 3/genética , Desmogleína 3/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Luciferases/química , Oligonucleotídeos , Proteínas/genética , Proteínas/imunologia
16.
Cancer Immunol Res ; 8(2): 230-245, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31852716

RESUMO

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.


Assuntos
Antígenos CD40/metabolismo , Antígeno CD47/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Interferon Tipo I/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/farmacologia , Imunidade Adaptativa , Animais , Ligante de CD40/genética , Ligante de CD40/imunologia , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Humanos , Imunidade Inata , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon Tipo I/metabolismo , Macaca fascicularis , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Neoplasias/patologia , Distribuição Aleatória , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
17.
J Infect Chemother ; 26(1): 115-118, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31591060

RESUMO

A 66-year-old man with a swollen right inguinal lymph node (LN) had pain on the lower side of the back. Computed tomography revealed bone disease in the back and swollen right inguinal LNs. Laboratory studies showed anemia and serum immunoglobulin G-lambda (IgG-λ) type monoclonal protein. The bone marrow contained 39.6% plasma cells. He was diagnosed with IgG-λ type multiple myeloma (MM). However, the pathological findings of the right inguinal LN were mixed cellular classical Hodgkin lymphoma (HL). The administration of melphalan, prednisone, and bortezomib (MPB) was started for MM; however, swelling in the right inguinal LN increased. After three cycles of MPB, the administration of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) was started for HL. However, HL was refractory to ABVD. Pancytopenia subsequently progressed and rapid swelling occurred in his LNs. He died 7 months after diagnosis. Multiple myeloma was diagnosed, based on the typical symptoms, although the pathological findings of the LN indicated a diagnosis of HL. We analyzed the molecular relationship between MM and HL cells using a direct sequencing method. The sequencing results demonstrated that the variable-diversity-joining (VDJ) region of the IgH gene was identified with 94.4% of IGLV3-32*01 in the bone marrow sample at diagnosis. Furthermore, clonotypic IgH sequence was identified in CD30-positive cells from the LN. These results suggested that the clonal HL cells were derived from the same source as the clonal MM cells and demonstrated that MM and HL in this patient may have originated from the same B cell progenitor.


Assuntos
Doença de Hodgkin , Cadeias lambda de Imunoglobulina/genética , Mieloma Múltiplo , Idoso , Dor nas Costas , Medula Óssea/patologia , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/genética , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfonodos/patologia , Masculino , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Pele/patologia , Tomografia Computadorizada por Raios X , Éxons VDJ/genética
18.
J Neuroimmunol ; 339: 577139, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31864140

RESUMO

To clarify the immunogenetic background of patients with immunoglobulin G (IgG)4 anti-neurofascin 155 (NF155) antibody-positive chronic inflammatory demyelinating polyneuropathy (CIDP), we genotyped the extended human leukocyte antigen (HLA) haplotypes in 22 Japanese patients with this disorder and compared them with those of healthy Japanese controls. All IgG4 anti-NF155 antibody-positive CIDP patients exclusively carried either HLA-DRB1*15:01-DRB5*01:01-DQA1*01:02-DQB1*06:02 or -(A*24:02)-B*52:01-C*12:02-DRB1*15:02-DRB5*01:02-DQA1*01:03-DQB1*06:01, resulting in significantly increased HLA-DRB1*15, -DRB1*15:01, -DQB1*06:01/06:02, -DQB1*06:02, and -DRB1*15:01-DQB1*06:02 frequencies compared with healthy Japanese controls. These findings indicate the involvement of specific HLA class II molecules in the pathomechanisms of IgG4 anti-NF155 antibody-positive CIDP.


Assuntos
Autoanticorpos/genética , Moléculas de Adesão Celular/genética , Antígenos HLA/genética , Haplótipos/genética , Imunoglobulina G/genética , Fatores de Crescimento Neural/genética , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/genética , Adolescente , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Autoanticorpos/sangue , Moléculas de Adesão Celular/sangue , Feminino , Antígenos HLA/sangue , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/sangue , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/sangue , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/diagnóstico , Adulto Jovem
19.
Protein Eng Des Sel ; 32(6): 277-288, 2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31868219

RESUMO

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.


Assuntos
Substituição de Aminoácidos , Imunoglobulina G/química , Imunoglobulina G/genética , Agregados Proteicos/genética , Engenharia de Proteínas , Eletricidade Estática , Imunoglobulina G/metabolismo , Modelos Moleculares , Conformação Proteica
20.
Nat Commun ; 10(1): 5389, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772165

RESUMO

The membrane-proximal external region (MPER) of HIV-1 envelope glycoprotein (Env) can be targeted by neutralizing antibodies of exceptional breadth. MPER antibodies usually have long, hydrophobic CDRH3s, lack activity as inferred germline precursors, are often from the minor IgG3 subclass, and some are polyreactive, such as 4E10. Here we describe an MPER broadly neutralizing antibody from the major IgG1 subclass, PGZL1, which shares germline V/D-region genes with 4E10, has a shorter CDRH3, and is less polyreactive. A recombinant sublineage variant pan-neutralizes a 130-isolate panel at 1.4 µg/ml (IC50). Notably, a germline revertant with mature CDR3s neutralizes 12% of viruses and still binds MPER after DJ reversion. Crystal structures of lipid-bound PGZL1 variants and cryo-EM reconstruction of an Env-PGZL1 complex reveal how these antibodies recognize MPER and viral membrane. Discovery of common genetic and structural elements among MPER antibodies from different patients suggests that such antibodies could be elicited using carefully designed immunogens.


Assuntos
Anticorpos Neutralizantes/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Farmacorresistência Viral/genética , Epitopos , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Conformação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA