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1.
Sci Transl Med ; 16(729): eadi1572, 2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38198572

RESUMO

CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.


Assuntos
Hepatite B Crônica , Interleucina-2 , Humanos , Animais , Camundongos , Vírus da Hepatite B , Linfócitos T CD8-Positivos , Hepatite B Crônica/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêutico
2.
Nat Med ; 24(7): 1005-1014, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29942088

RESUMO

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2-F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.


Assuntos
Anticorpos/química , Anticorpos/farmacologia , Interleucina-2/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Anticorpos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoterapia , Cinética , Camundongos Endogâmicos C57BL , Modelos Moleculares , Muromegalovirus/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Regulação para Cima/efeitos dos fármacos
3.
Protein Sci ; 15(4): 825-36, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16600968

RESUMO

A bacterial display methodology was developed for N- and C-terminal display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) display was directly compared to insertional fusion display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, bacterial display yielded the well-characterized HP(Q)/(M) motif obtained previously using several alternative peptide display systems, as well as three additional motifs (L(I)/(V) CQNVCY, CGWMY(F)/(Y)xEC, ERCWYVMHWPCNA). Using CPX display, a very high affinity streptavidin-binding peptide was isolated having a dissociation rate constant k(off) = 0.002sec(-1) even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N-terminal display yields sequences with greater diversity, affinity, and modularity. CPX bacterial display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.


Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Hidrolases/isolamento & purificação , Ligantes , Proteínas de Membrana/química , Biblioteca de Peptídeos , Peptídeos/química , Marcadores de Afinidade , Sequência de Aminoácidos , Afinidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo
4.
Protein Eng Des Sel ; 17(10): 731-9, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15531628

RESUMO

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.


Assuntos
Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Sequência Consenso , Escherichia coli/genética , Humanos , Técnicas In Vitro , Cinética , Ligantes , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
5.
Biotechnol Prog ; 20(3): 963-7, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15176905

RESUMO

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.


Assuntos
Antozoários/genética , Antozoários/metabolismo , Citometria de Fluxo/métodos , Biblioteca Gênica , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Sequência de Aminoácidos , Animais , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos
6.
Sci Transl Med ; 5(207): 207ra144, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24132639

RESUMO

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.


Assuntos
Anticorpos Antineoplásicos/uso terapêutico , Receptores ErbB/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pró-Fármacos/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cetuximab , Humanos , Imuno-Histoquímica , Macaca fascicularis , Camundongos , Camundongos Nus , Pró-Fármacos/toxicidade , Pele/efeitos dos fármacos , Pele/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Anal Chem ; 79(5): 2174-8, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17253874

RESUMO

The capability to screen molecular libraries using disposable microfluidic devices provides the potential to simplify and automate reagent generation and to develop integrated bioanalytical systems for clinical diagnostics. Here, antibody epitopes were mapped using a disposable microfluidic device to screen a combinatorial peptide library composed of 5 x 108 members displayed on bacterial cells. On-chip library screening was achieved in a two-stage, continuous-flow microfluidic sorter that separates antibody-binding target cells captured on microspheres through dielectrophoretic funneling. The antibody fingerprints identified were comparable to those obtained using state-of-the-art commercial cell sorting instrumentation.


Assuntos
Anticorpos Monoclonais/química , Mapeamento de Epitopos/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Biblioteca de Peptídeos , Sequência de Aminoácidos , Escherichia coli/genética , Dados de Sequência Molecular
9.
J Am Chem Soc ; 127(45): 15749-55, 2005 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-16277517

RESUMO

We introduce a novel method of inorganic synthesis using the catalytic and structure-directing properties of the demosponge enzyme silicatein-alpha. Recombinant silicatein-alpha was displayed at the surface of Escherichia coli cells by fusion to outer membrane protein A and used to biocatalytically direct the formation of layered and amorphous titanium phosphates from a small water-soluble precursor at near-neutral pH at 16 degrees C. Synthesis of titanium phosphates, with potential applications in catalysis and separation technology, previously has required prolonged reactions with phosphoric acid at elevated temperatures. Additionally, we use library screening to isolate a 15-mer with affinity toward the silicatein active site (Kd ca. 50 nM) and introduce this new approach to demonstrate the success of our display strategy. Considering our previous findings with native silicatein filaments, we suggest that this scalable, efficient, cell-based system may have a broad utility for the synthesis of a range of structured metallophosphates and other inorganic materials.


Assuntos
Catepsinas/genética , Catepsinas/metabolismo , Titânio/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Catepsinas/análise , Escherichia coli/química , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Sondas Moleculares/análise , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Titânio/metabolismo
10.
Proc Natl Acad Sci U S A ; 102(44): 15757-61, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16236724

RESUMO

Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery.


Assuntos
Separação Celular/métodos , Eletroforese em Microchip/métodos , Marcadores de Afinidade , Animais , Anticorpos Monoclonais , Antígenos de Superfície/imunologia , Bactérias/citologia , Bactérias/isolamento & purificação , Separação Celular/instrumentação , Separação Celular/normas , Células Clonais/citologia , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/normas , Desenho de Equipamento , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Microesferas
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