RESUMEN
Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 µM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Fibronectinas/química , Fragmentos de Péptidos/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Receptores ErbB/metabolismo , Femenino , Humanos , Immunoblotting , Cinética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Panitumumab , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Receptor tyrosine kinases (RTK) remain an area of therapeutic interest because of their role in epithelial tumors, and experimental models specific to these targets are highly desirable. Chimeric receptors were prepared by in-frame fusion of the CD8 extracellular sequence with the cytoplasmic sequences of RTKs. A CD8HER2 fusion protein was shown to form disulfide-mediated homodimers and to transform fibroblasts and epithelial cells. CD8RTK fusion proteins transform rat kidney epithelial cells and impart phenotypes that may reflect signaling specificity inherent in the native receptors. Transgenic expression of CD8HER2 and CD8Met in mice resulted in the formation of salivary and mammary gland tumors. The transgenic tumors allow the derivation of allograft tumors and cell lines that are sensitive to inhibition by small molecule kinase inhibitors. This approach provides excellent cell and tumor models for the characterization of signaling properties of diverse RTKs and for the evaluation of rationally designed antagonists targeting these kinases.
Asunto(s)
Antígenos CD8/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Mamarias Animales/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Neoplasias de las Glándulas Salivales/genética , Animales , Western Blotting , Transformación Celular Neoplásica/genética , Dimerización , Modelos Animales de Enfermedad , Disulfuros/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/etiología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Plásmidos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Neoplasias de las Glándulas Salivales/etiología , TransfecciónRESUMEN
NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.