RESUMO
The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.
Assuntos
Miniproteínas Nó de Cistina/metabolismo , Fibronectinas/metabolismo , Glioblastoma/irrigação sanguínea , Proteínas Recombinantes/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Miniproteínas Nó de Cistina/química , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/uso terapêutico , Fibronectinas/genética , Corantes Fluorescentes/química , Corantes Fluorescentes/uso terapêutico , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Imagem Óptica , Biblioteca de Peptídeos , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. RESULTS: The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. CONCLUSIONS: These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Histidina/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Vírion/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/isolamento & purificação , Modelos Moleculares , Controle de Qualidade , Proteínas Recombinantes/isolamento & purificação , Estresse Fisiológico , Vírion/ultraestruturaRESUMO
Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy.
Assuntos
Antígeno CTLA-4/metabolismo , Ciclotídeos/genética , Ciclotídeos/farmacologia , Avidina/metabolismo , Antígeno CTLA-4/química , Ciclotídeos/química , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
As membrane proteins play an important role in a variety of life-threatening diseases, the development of therapeutic monoclonal antibodies against membrane proteins is of significant interest. Among many other requirements, the process of antibody drug development requires a set of tailor-made assays for the characterization of the antibodies and for monitoring their activity. Designing assays to characterize antibodies directed to membrane proteins is challenging, because the natural targets are often not available in a format that is compatible with a biochemical assay setup. Thus, alternatives that mimic the targeted membrane proteins are needed. In this study, we developed optimal peptidic mimotopes for the ELISA-based detection of the novel therapeutic antibody IMAB362 in biological samples. Initial hits were identified using phage display and these hits were optimized with the help of structure-activity relationship analysis on peptide microarrays. The optimized peptides showed binding constants in the low nanomolar to picomolar range, an improvement by a factor of up to 30 compared to the initial hits. The best mimotope (apparent KD = 0.15 nM) was successfully used for the ELISA-based quantification of IMAB362 in samples from a mouse pharmacokinetic study. The process described allows the rapid discovery of mimotopes for target proteins that are difficult to produce or handle, which can then be used in pre-clinical and clinical assays or for the purification of biological products.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/farmacocinética , Peptídeos/química , Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.
Assuntos
Proteína de Ligação ao Complemento C4b/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Multimerização Proteica , Eletricidade Estática , TermodinâmicaRESUMO
Here we describe the facile generation of tetravalent peptide conjugates via a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) using a cyclic peptide template as a versatile conjugation scaffold. This stable and rigid framework is a conformationally constrained cyclic beta-sheet decorated with spatially defined alkyne moieties that serve as selectively addressable coupling sites. The proposed method allows for the effective coupling of unprotected peptide monomers in water at room temperature within comparatively short reaction times. The resulting conjugates display the ligands in an oriented manner, thus allowing for multivalent interactions with given target molecules, which may contribute to enhanced affinity and specificity. In addition, the selected scaffold offers an orthogonal coupling site for the incorporation of fluorescent labels or radioligands.
Assuntos
Alcinos/química , Azidas/química , Cobre/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Catálise , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/química , Glicina/análogos & derivados , Glicina/química , LigantesRESUMO
Microbodies are novel pharmacophoric entities which are derived from naturally occurring cystine-knot microproteins. They provide extremely stable scaffolds that can be engineered to high-affinity binding proteins. A peptide-grafting approach yielded specific ligands for human thrombopoietin receptor (TPO-R). Thrombopoietin (TPO) is the primary regulator of platelet production and acts by dimerization of its cognate receptor. Chemical cross linking of two anti TPO-R Microbodies resulted in highly potent TPO mimetics which are promising candidates for the treatment of TPO deficiencies. The approach demonstrates the high potential of dimeric Microbodies as synthetic receptor agonists.