RESUMEN
Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.
Asunto(s)
Dendrímeros/química , Nanopartículas/química , Polipropilenos/química , ARN Interferente Pequeño/química , Anticuerpos de Cadena Única/química , Línea Celular Tumoral , Endocitosis/genética , Endocitosis/fisiología , HumanosRESUMEN
Application of Toll-like receptor (TLR) agonists is a promising approach to treat cancer. In particular, nucleic acid-based TLR agonists such as short ssRNA and dsRNA molecules, which activate endosomal TLRs, can be delivered to tumors by use of nanoparticle delivery systems. However, such delivery systems bear unspecific side effects and poor pharmacokinetics. To overcome these limitations we developed a system for targeted delivery of a 50 bp dsRNA TLR3 agonist (Riboxxol) to treat PSCA-positive tumor cells, which consists of neutravidin conjugated to mono-biotinylated dsRNA and to humanized mono-biotinylated anti-PSCA single chain antibody derivative scFv(h-AM1)-BAP. The assembly of the components resulted in the formation of nanoparticle-like immunoconjugates designated Rapid Inducer of Cellular Inflammation and Apoptosis (RICIA). Anti-PSCA-RICIA exclusively delivered Riboxxol to PSCA-positive tumor cells as well as subcutaneous tumors. Uptake of anti-PSCA-RICIA induced a type I-interferon response and apoptosis in HEK-BluehTLR3/PSCA reporter cells and PSCA-positive HT1376 bladder cancer cells in vitro. No such effects were observed when using RICIA coupled to an unspecific control antibody or when using Riboxxol alone. Treatment of HT1376 xenografts in immune-deficient hosts with targeted delivery of TLR3 agonist did not induce adverse effects and only modestly inhibited tumor growth when compared to controls. These results suggest promising activation of innate immune response and apoptosis upon selective delivery of TLR3 agonists in tumor cells. Yet, further studies using syngeneic and orthotopic tumor models are needed to fully exploit the potential of RICIA immunoconjugates.
Asunto(s)
Apoptosis/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Inmunoconjugados , Interferón Tipo I/metabolismo , Nanopartículas , Proteínas de Neoplasias/agonistas , Neoplasias Experimentales , Anticuerpos de Cadena Única , Receptor Toll-Like 3/agonistas , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Ratones , Ratones Desnudos , Nanopartículas/química , Nanopartículas/uso terapéutico , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacología , Receptor Toll-Like 3/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In pretargeted radio-immunotherapy, the gradual administration of a non-radioactive tumor antigen-addressing antibody-construct and the subsequent application of a radioactive labeled, low molecular weight substance enable a highly effective and selective targeting of tumor tissue. We evaluated this concept in prostate stem cell antigen (PSCA)-positive cancers using the antigen-specific, biotinylated single chain antibody scFv(AM1)-P-BAP conjugated with tetrameric neutravidin. To visualize the systemic biodistribution, a radiolabeled biotin was injected to interact with scFv(AM1)-P-BAP/neutravidin conjugate. Biotin derivatives conjugated with different chelators for complexation of radioactive metal ions and a polyethylene glycol linker (n = 45) were successfully synthesized and evaluated in vitro and in a mouse xenograft model. In vivo, the scFv(AM1)-P-BAP showed highly PSCA-specific tumor retention with a PSCA+ tumor/PSCA- tumor accumulation ratio of ten. PEGylation of radiolabeled biotin resulted in lower liver uptake improving the tumor to background ratio.