RESUMEN
Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZTM assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody® therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions.
Asunto(s)
Imagen Molecular/métodos , Sondas Moleculares/metabolismo , Neoplasias/diagnóstico , Péptido Hidrolasas/análisis , Profármacos/metabolismo , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Microscopía Fluorescente , Sondas Moleculares/genética , Sondas Moleculares/farmacología , Neoplasias/patología , Péptido Hidrolasas/metabolismo , Profármacos/farmacología , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Profármacos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Unión Competitiva , Línea Celular Tumoral , Cetuximab/química , Cetuximab/metabolismo , Cetuximab/farmacología , Diagnóstico por Imagen/métodos , Receptores ErbB/metabolismo , Femenino , Colorantes Fluorescentes/química , Humanos , Neoplasias Pulmonares/metabolismo , Ratones Desnudos , Péptido Hidrolasas/metabolismo , Profármacos/química , Profármacos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Succinimidas/química , Factores de TiempoRESUMEN
Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.