RESUMO
We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Genes erbB-2 , Linhagem Celular Tumoral , Feminino , Genes erbB-2/efeitos dos fármacos , Humanos , Imunoterapia , Estrutura MolecularRESUMO
The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.
Assuntos
Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Engenharia Genética , Espaço Intracelular/metabolismo , Nanoestruturas , Neoplasias da Próstata/patologia , Proteínas/metabolismo , Sequência de Aminoácidos , Bacteriófago M13/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Dados de Sequência Molecular , Neoplasias da Próstata/metabolismo , Proteínas/genéticaRESUMO
Alanine scanning mutagenesis of a recently reported prostate cancer cell-selective Protein Transduction Domain (PTD) was used to assess the specific contribution each residue plays in cell uptake efficiency and cell-selectivity. These studies resulted in the identification of two key residues. Extensive mutagenesis at these key residues generated multiple mutants with significantly improved uptake efficiency and cell-selectivity profiles for targeted cells. The best mutant exhibits ~19-fold better uptake efficiency and ~4-fold improved cell-selectivity for a human prostate cancer cell line. In addition, while the native PTD sequence was capable of delivering functional fluorescent protein to the interior of a prostate cancer cells, only modest functional enzyme delivery was achieved. In contrast, the most potent mutant was able to deliver large quantities of a functional enzyme to the interior of human prostate cancer cells. Taken together, the research described herein has significantly improved the efficiency, cell-selectivity, and functional utility of a prostate cancer PTD.
Assuntos
Alanina/genética , Neoplasias da Próstata/genética , Estrutura Terciária de Proteína/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Masculino , Mutagênese , Neoplasias da Próstata/patologia , Transdução GenéticaRESUMO
Protein transduction domains (PTDs) are reagents that facilitate the delivery of diverse cargo to the interior of mammalian cells. We identified a PTD called "Ypep" (N-YTFGLKTSFNVQ-C), with cell penetration selectivity and potency profiles that are tightly controlled by multivalency effects. Pentavalent display of Ypep on M13 bacteriophage enables selective uptake of this phage in PC-3 human prostate cancer cells at low picomolar concentration and in the presence of human blood. All Ypep-dependent delivery is nontoxic and proceeds through energy-dependent endocytosis. Collectively, our results establish Ypep-displaying phage as a cell-penetrating platform with selectivity and potency profiles that compare to, or exceed, antibodies and their fragments. Our findings may have broader implications on the design of PTD technologies generated from phage display, as well as the use of Ypep-displaying phage as a prostate cancer cell-selective delivery platform.
Assuntos
Descoberta de Drogas , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Oligopeptídeos/química , Biblioteca de Peptídeos , Transporte Proteico/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
We report that N-4-nosyl-3-phenyloxaziridine is an effective terminal oxidant for copper(II)-catalyzed oxyamination recently developed in our labs. This oxaziridine can be prepared on multi-gram scale and is easily purified by recrystallization. The products of oxyamination using this oxaziridine bear protecting groups that can be readily removed in high yields under mild conditions.
