Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 109
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 21(8)2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32340383

RESUMO

The use of biotherapeutics for the treatment of diseases of the central nervous system (CNS) is typically impeded by insufficient transport across the blood-brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer's disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagement was confirmed in a biosensor experiment and the affinity for murine TfR was determined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.

2.
Int J Mol Sci ; 21(6)2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183096

RESUMO

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)3-ZHER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)3-ZHER3-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [57Co]Co (surrogate for 55Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [57Co]Co-(HE)3-ZHER3-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA 3 h pi, [57Co]Co-(HE)3-ZHER3-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [57Co]Co-chelator complex influenced the uptake in tumors and normal tissue. [57Co]Co-(HE)3-ZHER3-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [57Co]Co-(HE)3-ZHER3-DOTA improved imaging contrast compared to early-time-point imaging with [68Ga]Ga-(HE)3-ZHER3-NODAGA.

3.
Int J Mol Sci ; 21(4)2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32075258

RESUMO

Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)3-ZHER3:08698-DOTAGA affibody molecule with non-residualizing [125I]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA was compared side-by-side with [111In]In-(HE)3-ZHER3:08698-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24 h pi showed faster clearance of the [125I]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 ± 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [125I]I-PIB label. In conclusion, the use of non-residualizing [125I]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.

4.
Rev Sci Instrum ; 90(11): 113301, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31779428

RESUMO

We have devised an experimental method and apparatus for the simultaneous nondestructive determination of the absolute ion number, ion kinetic energy, and length of bunches of charged particles. We have built and operated a corresponding electronic detector that is based on induced charges and their subsequent low-noise amplification at cryogenic temperatures. We have performed measurements with bunches of low-energy highly charged ions from an electron-beam ion source that show the capability of the methods and their implementation. We discuss requirements for, and applications of, such detectors with a particular view on the obtainable information and their sensitivity.

5.
Sci Rep ; 9(1): 17710, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776413

RESUMO

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated 68Ga-labeled anti-HER3 affibody molecules (HE)3-ZHER3-DOTA and (HE)3-ZHER3-DOTAGA with previously reported [68Ga]Ga-(HE)3-ZHER3-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)3-ZHER3-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [68Ga]Ga-(HE)3-ZHER3-NODAGA. Presence of the negatively charged 68Ga-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [68Ga]Ga-(HE)3-ZHER3-DOTAGA and [68Ga]Ga-(HE)3-ZHER3-NODAGA had similar tumor-to-liver ratios, but [68Ga]Ga-(HE)3-ZHER3-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [68Ga]Ga-(HE)3-ZHER3-NODAGA remains the favorable variant for PET imaging of HER3 expression.

6.
Sci Rep ; 9(1): 6779, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043683

RESUMO

Human epidermal growth factor receptor type 3 (HER3) plays a crucial role in the progression of many cancer types. In vivo radionuclide imaging could be a reliable method for repetitive detection of HER3-expression in tumors. The main challenge of HER3-imaging is the low expression in tumors together with endogenous receptor expression in normal tissues, particularly the liver. A HER3-targeting affibody molecule labeled with radiocobalt via a NOTA chelator [57Co]Co-NOTA-Z08699 has demonstrated the most favorable biodistribution profile with the lowest unspecific hepatic uptake and high activity uptake in tumors. We hypothesized that specific uptake of labeled affibody monomer might be selectively blocked in the liver but not in tumors by a co-injection of non-labeled corresponding trivalent affibody (Z08699)3. Biodistribution of [57Co]Co-NOTA-Z08699 and [111In]In-DOTA-(Z08699)3 was studied in BxPC-3 xenografted mice. [57Co]Co-NOTA-Z08699 was co-injected with unlabeled trivalent affibody DOTA-(Z08699)3 at different monomer:trimer molar ratios. HER3-expression in xenografts was imaged using [57Co]Co-NOTA-Z08699 and [57Co]Co-NOTA-Z08699: DOTA-(Z08699)3. Hepatic activity uptake of [57Co]Co-NOTA-Z08699: DOTA-(Z08699)3 decreased with increasing monomer:trimer molar ratio. The tumor activity uptake and tumor-to-liver ratios were the highest for the 1:3 ratio. SPECT/CT images confirmed the biodistribution data. Imaging of HER3 expression can be improved by co-injection of a radiolabeled monomeric affibody-based imaging probe together with a trivalent affibody.

7.
Front Aging Neurosci ; 11: 64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30967771

RESUMO

Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid ß (anti-Aß) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of Aß-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted ZSYM73-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric ZSYM73 affibody for sequestering of monomeric Aß-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with ZSYM73-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric Aß, demonstrating a therapeutic potential for prevention of AD.

8.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832342

RESUMO

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.


Assuntos
Radioisótopos de Gálio/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-3/metabolismo , Acetatos/química , Animais , Linhagem Celular Tumoral , Quelantes/química , Compostos Heterocíclicos com 1 Anel/química , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Compostos Radiofarmacêuticos/química , Proteínas Recombinantes de Fusão/química , Distribuição Tecidual
9.
Sci Rep ; 9(1): 655, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679757

RESUMO

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z08698 and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged 111In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

10.
Oncol Rep ; 41(1): 534-542, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30320363

RESUMO

The epidermal growth factor receptor (EGFR) is often overexpressed during prostate cancer (PCa) progression towards androgen­independence after hormone therapy, but the overexpression is lower than in other types of cancers. Despite the low expression, EGFR has emerged as a promising therapeutic target for patients with castration­resistant PCa. Non­invasive methods for determination of EGFR expression in PCa can serve for patient stratification and therapy response monitoring. Radionuclide imaging probes based on affibody molecules (7 kDa) provide high contrast imaging of cancer­associated molecular targets. We hypothesized that the anti­EGFR affibody molecule DOTA­ZEGFR:2377 labeled with 55Co (positron­emitter, T1/2=17.5 h) would enable imaging of EGFR expression in PCa xenografts. The human PCa cell line DU­145 was used for in vitro and in vivo experiments and 57Co was used as a surrogate for 55Co in the present study. Binding of 57Co­DOTA­ZEGFR:2377 to EGFR­expressing xenografts was saturable with anti­EGFR monoclonal antibody cetuximab, which would motivate the use of this tracer for monitoring the receptor occupancy during treatment. A significant dose­dependent difference in radioactivity accumulation in tumors and normal organs was observed when the biodistribution was studied 3 h after the injection of 10 and 35 µg of 57Co­DOTA­ZEGFR:2377: At lower doses the tumor uptake was 2­fold higher although tumor­to­organ ratios were not altered. For clinically relevant organs for PCa, tumor­to­organ ratios increased with time, and at 24 h pi were 2.2±0.5 for colon, 7±2 for muscle, and 4.0±0.7 for bones. Small animal SPECT/CT images confirmed the capacity of radiocobalt labeled DOTA­ZEGFR:2377 to visualize EGFR expression in PCa. In conclusion, the present study demonstrated the feasibility of using the radiocobalt labeled anti­EGFR affibody conjugate ZEGFR:2377 as an imaging agent for in vivo visualization of low EGFR­expressing tumors, like PCa, and for monitoring of receptor occupancy during cetuximab therapy as well as the importance of optimal dosing in order to achieve higher sensitivity molecular imaging.


Assuntos
Isótopos do Cobalto/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular/métodos , Cintilografia/métodos , Distribuição Tecidual/fisiologia
11.
Biotechnol J ; 14(4): e1800359, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30179307

RESUMO

Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naïve affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 109 variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.


Assuntos
Adesinas de Escherichia coli/genética , Anticorpos Monoclonais/biossíntese , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Adesinas de Escherichia coli/biossíntese , Adesinas de Escherichia coli/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Escherichia coli/química , Escherichia coli/genética , Citometria de Fluxo , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química
12.
Cells ; 7(10)2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30314301

RESUMO

Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

13.
Theranostics ; 8(16): 4462-4476, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30214632

RESUMO

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key mediator of angiogenesis and therefore a promising therapeutic target in malignancies including glioblastoma multiforme (GBM). Molecular imaging of VEGFR2 expression may enable patient stratification for antiangiogenic therapy. The goal of the current study was to evaluate the capacity of the novel anti-VEGFR2 biparatopic affibody conjugate (ZVEGFR2-Bp2) for in vivo visualization of VEGFR2 expression in GBM. Methods: ZVEGFR2-Bp2 coupled to a NODAGA chelator was generated and radiolabeled with indium-111. The VEGFR2-expressing murine endothelial cell line MS1 was used to evaluate in vitro binding specificity and affinity, cellular processing and targeting specificity in mice. Further tumor targeting was studied in vivo in GL261 glioblastoma orthotopic tumors. Experimental imaging was performed. Results: [111In]In-NODAGA-ZVEGFR2-Bp2 bound specifically to VEGFR2 (KD=33±18 pM). VEGFR2-mediated accumulation was observed in liver, spleen and lungs. The tumor-to-organ ratios 2 h post injection for mice bearing MS1 tumors were approximately 11 for blood, 15 for muscles and 78 for brain. Intracranial GL261 glioblastoma was visualized using SPECT/CT. The activity uptake in tumors was significantly higher than in normal brain tissue. The tumor-to-cerebellum ratios after injection of 4 µg [111In]In-NODAGA-ZVEGFR2-Bp2 were significantly higher than the ratios observed for the 40 µg injected dose and for the non-VEGFR2 binding size-matched conjugate, demonstrating target specificity. Microautoradiography of cryosectioned CNS tissue was in good agreement with the SPECT/CT images. Conclusion: The anti-VEGFR2 affibody conjugate [111In]In-NODAGA-ZVEGFR2-Bp2 specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [111In]In-NODAGA-ZVEGFR2-Bp2 were higher compared to other VEGFR2 imaging probes. [111In]In-NODAGA-ZVEGFR2-Bp2 appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.


Assuntos
Células Endoteliais/química , Glioma/diagnóstico por imagem , Glioma/patologia , Imagem Molecular/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos/administração & dosagem , Linhagem Celular , Camundongos , Estudo de Prova de Conceito , Proteínas Recombinantes de Fusão/administração & dosagem , Sensibilidade e Especificidade
14.
Mol Pharm ; 15(8): 3394-3403, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29995421

RESUMO

Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER-targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor ErbB-3/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Neoplasias/mortalidade , Neoplasias/patologia , Neuregulina-1/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Rev Sci Instrum ; 88(9): 093903, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28964189

RESUMO

Shot noise measurements on atomic and molecular junctions provide rich information about the quantum transport properties of the junctions and on the inelastic scattering events taking place in the process. Dissipation at the nanoscale, a problem of central interest in nano-electronics, can be studied in its most explicit and simplified form. Here, we describe a measurement technique that permits extending previous noise measurements to a much higher frequency range, and to much higher bias voltage range, while maintaining a high accuracy in noise and conductance. We also demonstrate the advantages of having access to the spectral information for diagnostics.

16.
Appl Microbiol Biotechnol ; 101(23-24): 8293-8307, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28971248

RESUMO

Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and "food grade" species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.


Assuntos
Microbiologia de Alimentos/métodos , Engenharia de Proteínas/métodos , Staphylococcus/genética , Staphylococcus/metabolismo , Biotecnologia/métodos , Técnicas de Visualização da Superfície Celular/métodos , Fermentação , Staphylococcus/crescimento & desenvolvimento
17.
Int J Oncol ; 51(6): 1765-1774, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039474

RESUMO

The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-targeted therapies. Several HER3­targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-to-blood ratios significantly increased between 3 and 24 h p.i. (p<0.05). At 24 h p.i., tumour-to-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3­expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.


Assuntos
Radioisótopos de Cobalto , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/enzimologia , Compostos Radiofarmacêuticos , Receptor ErbB-3/análise , Receptor ErbB-3/biossíntese , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/enzimologia , Feminino , Compostos Heterocíclicos , Compostos Heterocíclicos com 1 Anel , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos
18.
Sci Rep ; 7(1): 5949, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729665

RESUMO

Protofibrils of the 42 amino acids long amyloid-ß peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer's disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aß42cc, which is a stable engineered mimic of wild type Aß42 protofibrils. Several binders were identified that bind Aß42cc protofibrils with low nanomolar affinities, and which also recognize wild type Aß42 protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aß42cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aß42 protofibrils.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Técnicas de Visualização da Superfície Celular , Cinética , Fragmentos de Peptídeos/química , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química
19.
Sci Rep ; 7(1): 5961, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729680

RESUMO

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with 57Co (T1/2 = 271.8 d), 55Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57Co was used in animal studies. Both 57Co-DOTA-ZEGFR:2377 and 68Ga-DOTA-ZEGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-ZEGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57Co-DOTA-ZEGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68Ga-DOTA-ZEGFR:2377. The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-ZEGFR:2377 and further development should concentrate on this radionuclide as a label.


Assuntos
Complexos de Coordenação/química , Receptores ErbB/metabolismo , Compostos Heterocíclicos com 1 Anel/química , Imageamento Tridimensional , Radioisótopos/química , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos Endogâmicos BALB C , Camundongos Nus , Tomografia por Emissão de Pósitrons , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Trends Biotechnol ; 35(8): 691-712, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28514998

RESUMO

Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.


Assuntos
Biotecnologia , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Biotecnologia/métodos , Biotecnologia/tendências , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Transdução de Sinais/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA