RESUMEN
A growing class of immunotherapeutics work by redirecting components of the immune system to recognize markers on the surface of cancer cells. However, such modalities will remain confined to a relatively small subgroup of patients because of the lack of universal targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidity of solid tumors to selectively graft cancer cells with immuno-engager epitopes. Our targeting approach is based on pHLIP, a unique peptide that selectively targets tumors in vivo by anchoring to cancer cell surfaces in a pH-dependent manner. We established that pHLIP-antigen conjugates trigger the recruitment of antibodies to the surface of cancer cells and induce cytotoxicity by peripheral blood mononuclear and engineered NK cells. These results indicate that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent problems associated with narrow windows of selectivity.
Asunto(s)
Epítopos/farmacología , Factores Inmunológicos/farmacología , Proteínas de la Membrana/farmacología , 2,4-Dinitrofenol/química , 2,4-Dinitrofenol/inmunología , 2,4-Dinitrofenol/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Epítopos/química , Epítopos/inmunología , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacología , Humanos , Concentración de Iones de Hidrógeno , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Inmunoterapia/métodos , Células Asesinas Naturales/efectos de los fármacos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Receptor protein tyrosine phosphatases (RPTPs) play critical regulatory roles in mammalian signal transduction. However, the structural basis for the regulation of their catalytic activity is not fully understood, and RPTPs are generally not therapeutically targetable. This knowledge gap is partially due to the lack of known natural ligands or selective agonists of RPTPs. Contrary to what is known from structure-function studies of receptor tyrosine kinases (RTKs), RPTP activities have been reported to be suppressed by dimerization, which may prevent RPTPs from accessing their RTK substrates. We report here that homodimerization of protein tyrosine phosphatase receptor J (PTPRJ, also known as DEP-1) is regulated by specific transmembrane (TM) residues. We found that disrupting these interactions destabilizes homodimerization of full-length PTPRJ in cells, reduces the phosphorylation of the known PTPRJ substrate epidermal growth factor receptor (EGFR) and of other downstream signaling effectors, antagonizes EGFR-driven cell phenotypes, and promotes substrate access. We demonstrate these observations in human cancer cells using mutational studies and identified a peptide that binds to the PTPRJ TM domain and represents the first example of an allosteric agonist of RPTPs. The results of our study provide fundamental structural and functional insights into how PTPRJ activity is tuned by TM interactions in cells. Our findings also open up opportunities for developing peptide-based agents that could be used as tools to probe RPTPs' signaling mechanisms or to manage cancers driven by RTK signaling.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Línea Celular Tumoral , Humanos , Immunoblotting , Fosforilación/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal/fisiología , Espectrometría de FluorescenciaRESUMEN
Overexpression and deregulation of the epidermal growth factor receptor (EGFR) are implicated in multiple human cancers and therefore are a focus for the development of therapeutics. Current strategies aimed at inhibiting EGFR activity include monoclonal antibodies and tyrosine kinase inhibitors. However, activating mutations severely limit the efficacy of these therapeutics. There is thus a growing need for novel methods to inhibit EGFR. One promising approach involves blocking the association of the cytoplasmic juxtamembrane (JM) domain of EGFR, which has been shown to be essential for receptor dimerization and kinase function. Here, we aim to improve the selectivity and efficacy of an EGFR JM peptide mimic by utilizing the pH(low) insertion peptide (pHLIP), a unique molecule that can selectively target cancer cells solely based on their extracellular acidity. This delivery strategy potentially allows for more selective targeting to tumors than current methods and for anchoring the peptide mimic to the cytoplasmic leaflet of the plasma membrane, increasing its local concentration and thus efficacy. We show that the conjugated construct is capable of inhibiting EGFR phosphorylation and downstream signaling and of inducing concentration- and pH-dependent toxicity in cervical cancer cells. We envision that this approach could be expanded to the modulation of other single-span membrane receptors whose activity is mediated by JM domains.