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1.
Nat Chem Biol ; 18(8): 850-858, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35654846

RESUMO

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.


Assuntos
Antígeno B7-H1 , Leucócitos Mononucleares , Comunicação Celular , Flavinas , Imunoterapia
2.
Bioconjug Chem ; 34(9): 1633-1644, 2023 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-37620302

RESUMO

Antibody-drug conjugates (ADCs) have garnered worldwide attention for disease treatment, as they possess high target specificity, a long half-life, and outstanding potency to kill or modulate the functions of targets. FDA approval of multiple ADCs for cancer therapy has generated a strong desire for novel conjugation strategies with high biocompatibility and controllable bioproperties. Herein, we present a bisecting glycan-bridged conjugation strategy that enables site-specific conjugation without the need for the oligosaccharide synthesis and genetic engineering of antibodies. Application of this method is demonstrated by conjugation of anti-HER2 human and mouse IgGs with a cytotoxic drug, monomethyl auristatin E. The glycan bridge showed outstanding stability, and the resulting ADCs eliminated HER2-expressing cancer cells effectively. Moreover, our strategy preserves the feasibility of glycan structure remodeling to fine-tune the immunogenicity and pharmacokinetic properties of ADCs through glycoengineering.


Assuntos
Anticorpos , Imunoconjugados , Humanos , Animais , Camundongos , Imunoconjugados/uso terapêutico , Engenharia Genética , Meia-Vida , Polissacarídeos
3.
Nat Chem Biol ; 6(5): 338-43, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20305658

RESUMO

Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.


Assuntos
Acetilglucosamina/química , Anticorpos Monoclonais/imunologia , Glicopeptídeos/imunologia , Acetilglucosamina/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação
4.
J Pept Sci ; 16(12): 716-22, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21104968

RESUMO

The membrane proximal external region (MPER) of gp41 abuts the viral membrane at the base of HIV-1 envelope glycoprotein spikes. The MPER is highly conserved and is rich in Trp and other lipophilic residues. The MPER is also required for the infection of host cells by HIV-1 and is the target of the broadly neutralizing antibodies, 4E10, 2F5, and Z13e1. These neutralizing antibodies are valuable tools for understanding relevant conformations of the MPER and for studying HIV-1 neutralization, but multiple approaches used to elicit MPER binding antibodies with similar neutralization properties have failed. Here we report our efforts to mimic the MPER using linear as well as constrained peptides. Unnatural amino acids were also introduced into the core epitope of 4E10 to probe requirements of antibody binding. Peptide analogs with C-terminal Api or Aib residues designed to be helical transmembrane (TM) domain surrogates exhibit enhanced binding to the 4E10 and Z13e1 antibodies. However, we find that placement of constrained amino acids at nonbinding sites within the core epitope significantly reduce binding. These results are relevant to an understanding of native MPER structure on HIV-1, and form a basis for a chemical synthesis approach to mimic MPER stricture and to construct an MPER-based vaccine.


Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/síntese química , Aminoácidos/química , Modelos Moleculares , Estrutura Molecular
5.
Chembiochem ; 10(3): 455-63, 2009 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-19145607

RESUMO

SYNTHETIC CANCER VACCINES: A number of fully synthetic vaccine candidates have been designed, chemically synthesized, and immunologically evaluated to establish a strategy to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides and to determine the importance of Toll-like receptor (TLR) engagement for antigenic responses against these compounds.Epithelial cancer cells often overexpress mucins that are aberrantly glycosylated. Although it has been realized that these compounds offer exciting opportunities for the development of immunotherapy for cancer, their use is hampered by the low antigenicity of classical immunogens composed of a glycopeptide derived from a mucin conjugated to a foreign carrier protein. We have designed, chemically synthesized, and immunologically evaluated a number of fully synthetic vaccine candidates to establish a strategy to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. The compounds were also designed to allow study of the importance of Toll-like receptor (TLR) engagement for these antigenic responses in detail. We have found that covalent attachment of a TLR2 agonist, a promiscuous peptide T-helper epitope, and a tumor-associated glycopeptide gives a compound (1) that elicits in mice exceptionally high titers of IgG antibodies that recognize MCF7 cancer cells expressing the tumor-associated carbohydrate. Immunizations with glycolipopeptide 2, which contains lipidated amino acids instead of a TLR2 ligand, gave significantly lower titers of IgG antibodies; this demonstrates that TLR engagement is critical for optimum antigenic responses. Although mixtures of compound 2 with Pam(3)CysSK(4) (3) or monophosphoryl lipid A (4) elicited titers of IgG antibodies similar to those seen with 1, the resulting antisera had impaired ability to recognize cancer cells. It was also found that covalent linkage of the helper T-epitope to the B-epitope is essential, probably because internalization of the helper T-epitope by B-cells requires assistance of the B-epitope. The results presented here show that synthetic vaccine development is amenable to structure-activity relationship studies for successful optimization of carbohydrate-based cancer vaccines.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer , Proteínas Recombinantes/imunologia , Receptores Toll-Like/imunologia , Sequência de Aminoácidos , Animais , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/genética , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Linhagem Celular , Epitopos , Feminino , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estrutura Molecular , Mucina-1/genética , Mucina-1/imunologia , Engenharia de Proteínas , Proteínas Recombinantes/genética , Receptores Toll-Like/genética
6.
Angew Chem Int Ed Engl ; 48(10): 1775-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19072969

RESUMO

Changing employment: Receptor 1 binds beta-N-acetylglucosaminyl (beta-GlcNAc) up to 100 times more strongly than it does glucose. This synthetic lectin shows affinities similar to wheat germ agglutinin (WGA), a natural lectin used to bind GlcNAc. Remarkably, 1 is more selective than WGA. It favors especially the glycoside unit in glycopeptide 2, a model of the serine-O-GlcNAc posttranslational protein modification.


Assuntos
Acetilglucosamina/química , Lectinas/química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Carboidratos/química , Cinética , Lectinas/síntese química , Lectinas/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Processamento de Proteína Pós-Traducional , Aglutininas do Germe de Trigo/metabolismo
7.
Org Lett ; 8(25): 5785-8, 2006 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-17134272

RESUMO

Although native chemical ligation (NCL) is emerging as a powerful method for the assembly of (glyco)peptide building blocks, its applicability is reduced when peptide segments are poorly soluble in aqueous buffer. We have found that incorporating reactants in liposomes allows NCL of lipophilic peptides and lipopeptides. Furthermore, the reaction rates of liposome-mediated NCL are higher than traditional reaction conditions resulting in improved yields. [reaction: see text]


Assuntos
Glicopeptídeos/síntese química , Lipossomos/química , Sequência de Aminoácidos , Soluções Tampão , Vacinas Anticâncer/química , Epitopos , Glicopeptídeos/química , Indicadores e Reagentes , Dados de Sequência Molecular , Mucina-1/química , Solubilidade
8.
PLoS One ; 10(5): e0125581, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25938510

RESUMO

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Infecções por HIV/imunologia , Ensaios de Triagem em Larga Escala/métodos , Análise Serial de Proteínas/métodos , Sequência de Aminoácidos , Especificidade de Anticorpos/imunologia , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/sangue , HIV-1/imunologia , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/química , Polissacarídeos/imunologia , Alinhamento de Sequência
9.
ACS Nano ; 7(5): 3778-96, 2013 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-23710591

RESUMO

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide's ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.


Assuntos
Peptídeos Penetradores de Células/química , Citosol/metabolismo , Portadores de Fármacos/química , Proteínas Ligantes de Maltose/metabolismo , Pontos Quânticos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Peptídeos Penetradores de Células/metabolismo , Embrião de Galinha , Portadores de Fármacos/metabolismo , Endossomos/metabolismo , Humanos , Dados de Sequência Molecular
10.
Org Lett ; 13(11): 2822-5, 2011 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-21553819

RESUMO

Triazole tethers have been explored for stabilization of secondary structures in peptides. Despite the utility of this approach, cyclization efficiency in complex peptides remains a significant challenge. A robust, on-resin protocol for side chain to side chain macrocyclization by CuAAC is described. This protocol was applied to the synthesis of a series of 21 amino acid helical peptides presenting a binding dipeptide motif from the membrane proximal external region (MPER) of HIV-1 gp41.


Assuntos
Proteína gp41 do Envelope de HIV/química , Fragmentos de Peptídeos/química , Peptídeos/química , Triazóis/síntese química , Sequência de Aminoácidos , Dicroísmo Circular , Cobre/química , Ciclização , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Estrutura Molecular , Fragmentos de Peptídeos/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Triazóis/química
12.
Nat Chem Biol ; 3(10): 663-7, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17767155

RESUMO

The overexpression of saccharides such as Globo-H, Lewis(Y) and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development have been complicated by difficulties in eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Sequência de Aminoácidos , Animais , Antígenos Glicosídicos Associados a Tumores/química , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/uso terapêutico , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Epitopos/imunologia , Epitopos/metabolismo , Glicopeptídeos/síntese química , Imunoglobulina G/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Regulação para Cima
13.
Glycobiology ; 16(8): 113R-136R, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16675547

RESUMO

This review describes the recent advances in the field of glycopeptide and small glycoprotein synthesis. The strategies covered include chemical and chemoenzymatic synthesis, native chemical ligation (NCL), and expressed chemical ligation. The importance of glycopeptide synthesis is exemplified by giving the reader an overview of how versatile and important these well-defined glycopeptides are as tools in glycobiology.


Assuntos
Biologia , Glicopeptídeos/síntese química , Glicoproteínas/síntese química , Modelos Químicos , Oligossacarídeos/síntese química
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