RESUMEN
The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.
Asunto(s)
Neoplasias , Péptidos , Animales , Apoptosis , Línea Celular , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Péptidos/químicaRESUMEN
Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein-protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.
Asunto(s)
Interleucina-23/antagonistas & inhibidores , Péptidos/química , Secuencia de Aminoácidos , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/uso terapéutico , Psoriasis/prevención & controlAsunto(s)
Antígenos/inmunología , Biotina , Ensayo de Inmunoadsorción Enzimática/métodos , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Biblioteca de Péptidos , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Indicadores y Reactivos , EstreptavidinaRESUMEN
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de la Metaloproteinasa de la Matriz , Neovascularización Patológica/prevención & control , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/patología , Línea Celular Tumoral , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Invasividad Neoplásica/patología , Transfección , Trasplante Heterólogo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.
Asunto(s)
Antifibrinolíticos/farmacología , Antineoplásicos/farmacología , Polietilenglicoles/farmacología , Animales , Antifibrinolíticos/farmacocinética , Antineoplásicos/farmacocinética , Coagulación Sanguínea/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/antagonistas & inhibidores , Femenino , Hemostasis/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos BALB C , Conejos , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.