RESUMO
Recombinant immunotoxins are extremely promising agents for the targeted therapy of tumors with a certain molecular profile. In this work, we studied the properties of a new recombinant HER2-specific immunotoxin composed of the scFv antibody and a fragment of Pseudomonas exotoxin A (4D5scFv-PE40). High affinity of the immunotoxin for the HER2 tumor marker, its selective cytotoxicity against HER2-overexpressing cells, and its storage stability were demonstrated. The 50% inhibitory concentration (IC50) of the 4D5scFv-PE40 immunotoxin for HER2-overexpressing cancer cells was 2.5-3 orders of magnitude lower compared to that for CHO cells not expressing this tumor marker and was 2.5-3 orders of magnitude lower than IC50 of free PE40 for HER2-overexpressing cancer cells. These findings provide a basis for expecting in the long run high therapeutic index values of the 4D5scFv-PE40 immunotoxin for its use in vivo.
RESUMO
Targeted delivery of semiconductor quantum dots (Q Ds) to tumors overexpressing HER2 cancer marker has been. demonstrated on immunocompromised mice bearing human breast cancer xenografts. To obtain targeted QDs complexes we applied the approach based on the use of protein adaptor system, RNAase barnase and its inhibitor barstar. Specific binding to target cancer marker was achieved through bivalent fusion protein containing two fragments of4D5scFv recombinant antibody and a fragment of barnase. QDs were conjugated to barstar, and final assembly of targeted complexes was obtained through non-covalent specific interaction of barstar, attached to QD, and barnase, that is part of the recombinant targeting protein. The efficient delivery of QDs to HER2-expressing tumor demonstrates the possibilities and prospects of the approach for targeted delivery of nanoparticles to cancer cells in vivo as the way to improve the efficiency of diagnosis and promote development of therapies based on the use of nanoparticles.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Marcação de Genes , Neoplasias Mamárias Experimentais/genética , Pontos Quânticos/administração & dosagem , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pontos Quânticos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ribonucleases/administração & dosagem , Ribonucleases/química , Ribonucleases/genéticaRESUMO
The novel HER2/neu-specific recombinant immunotoxin 4D5scFv-PE40 consisting of 4D5scFv antibody (targeting module) and Pseudomonas exotoxin A fragment (effector module) combined in a single polypeptide chain via a flexible linker has been expressed and purified. This immunotoxin conserves specificity and affinity that are characteristics of the parental antibody 4D5scFv and exhibits selective and strong cytotoxic effect against cancer cells overexpressing HER2/neu receptor. The results of the experiments both in vitro (in cell cultures) and in vivo (in tumor-bearing animals) demonstrate high potential of 4D5scFv-PE40 for targeted therapy of tumors overexpressing HER2/neu.
Assuntos
Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antineoplásicos/química , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Exotoxinas/genética , Feminino , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaAssuntos
Acroleína/química , Compostos de Cádmio/química , Corantes Fluorescentes/química , Imagem Molecular/métodos , Polímeros/química , Pontos Quânticos , Compostos de Selênio/química , Semicondutores , Sulfetos/química , Compostos de Zinco/química , Aglutinação , Células HeLa , Humanos , Látex/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Semiconductor quantum dots (QDs) are a new class of fluorophores with unique physical and chemical properties, which allow to appreciably expand the possibilities for the current methods of fluorescent imaging and optical diagnostics. Here we discuss the prospects of QD application for molecular diagnostics of tumors ranging from cancer-specific marker detection on microplates to non-invasive tumor imagingin vivo. We also point out the essential problems that require resolution in order to clinically promote QD, and we indicate innovative approaches to oncology which are implementable using QD.
Assuntos
Imagem Molecular/métodos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Pontos Quânticos , Anticorpos/genética , Anticorpos/metabolismo , Compostos de Cádmio/química , Compostos de Cádmio/metabolismo , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Regulação Neoplásica da Expressão Gênica , Engenharia Genética , Humanos , Neoplasias Ovarianas/genética , Receptor ErbB-2/metabolismo , Compostos de Selênio/química , Compostos de Selênio/metabolismo , Sulfetos/química , Sulfetos/metabolismoRESUMO
The uptake of quantum dots (QD-5 nm particles of CdSe/ZnS-mercaptoacetic acid) by human neutrophilic granulocytes was studied using the methods of scanning laser and scanning probe microscopy. The results show that the neutrophilic granulocytes may be subdivided into three subpopulations: (1) the cells with no uptake of QD (10.0 +/- 2.0%); (2) cells that accumulate QD in their volume (28.0 +/- 1.9%), and (3) cells, surrounded by a halo of QD (59.0 +/- 2.2%). The dispersion of these characteristics may suggest the differences in neutrophilic granulocyte plasma membrane permeability.
