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1.
Clin Cancer Res ; 28(15): 3387-3399, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35121624

RESUMO

PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.


Assuntos
Antígeno B7-H1 , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral
2.
Biomol NMR Assign ; 10(1): 41-3, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26280529

RESUMO

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein-protein recognition events mediated by conserved motifs. The sequence-specific (1)H, (15)N, and (13)C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C's complex network of molecular interactions with the autophagic machinery by NMR spectroscopy.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Isótopos de Carbono , Humanos , Isótopos de Nitrogênio , Trítio
3.
PLoS One ; 8(9): e72272, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039747

RESUMO

In this work, we exploited a method that uses polytopic membrane proteins as targets for phage display selections. Membrane proteins represent the largest class of drug targets and drug discovery is mostly based on the identification of ligands binding to target molecules. The screening of a phage display library for ligands against membrane proteins is typically hindered by the requirement of these proteins for a membrane environment, which is necessary to retain correct folding and epitope formation. Especially in proteins with multiple transmembrane domains, epitopes often are non-linear and consist of a combination of loops between transmembrane stretches of the proteins. Here, we have used bacteriorhodopsin (bR) as a model of polytopic membrane protein, assembled into nanoscale phospholipid bilayers, so called nanodiscs, to screen a phage display library for potential ligands. Nanodiscs provide a native-like environment to membrane proteins and thus selection of ligands can take place in a near physiological state. Screening a 12-mer phage display peptide library against bR nanodiscs led to the isolation of phage clones binding specifically to bR. We were further able to identify the binding site of selected phage clones proving that the clones bind to extramembranous, non-linear epitopes of bR. Thus, nanodiscs provide a suitable and general tool that allows screening of a phage display library against membrane proteins in a near native environment.


Assuntos
Bacteriorodopsinas/química , Dimiristoilfosfatidilcolina/química , Nanopartículas/química , Sequência de Aminoácidos , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/química , Ligantes , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Ligação Proteica
4.
FEMS Microbiol Lett ; 319(1): 26-33, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21401713

RESUMO

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Regulação Enzimológica da Expressão Gênica , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Peptídeo Sintases/química , Peptídeo Sintases/genética , Policetídeo Sintases/química , Policetídeo Sintases/genética , Estrutura Terciária de Proteína , Piridonas/metabolismo , Streptomyces/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética
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