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1.
Life Sci ; 257: 118052, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634431

RESUMO

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.


Assuntos
Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Receptores de Fator Estimulador de Colônias de Granulócitos/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos , Anticorpos Monoclonais/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Neovascularização Patológica/tratamento farmacológico , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única
2.
Biochemistry ; 41(20): 6422-31, 2002 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-12009905

RESUMO

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.


Assuntos
Fator Estimulador de Colônias de Granulócitos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos/química , Precipitação Química , Dicroísmo Circular , Deutério/química , Dimerização , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Humanos , Hidrogênio/química , Cinética , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes , Soluções , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Sacarose/química , Propriedades de Superfície , Termodinâmica
3.
Biochem Biophys Res Commun ; 201(3): 1396-400, 1994 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-7517669

RESUMO

Schemes of the four-helix bundle surfaces of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, interferon-beta, -gamma and growth hormone were designed. All cytokines appeared to have the structurally similar "holes" on the surfaces. They were suggested to serve as a part of the main receptor-binding sites.


Assuntos
Citocinas/ultraestrutura , Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Hormônio do Crescimento/química , Interferon beta/ultraestrutura , Interferon gama/ultraestrutura , Interleucina-2/química , Interleucina-5/química , Fator Estimulador de Colônias de Macrófagos/ultraestrutura , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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