RESUMEN
Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.
Asunto(s)
Alginatos/química , Hidrogeles/química , Nanopartículas del Metal/química , Línea Celular Tumoral , Liberación de Fármacos , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Oro/química , Humanos , Unión ProteicaRESUMEN
Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/inmunología , Anticuerpos Monoclonales/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias de la Próstata/tratamiento farmacológico , Antibióticos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Antineoplásicos Fitogénicos/administración & dosificación , Comunicación Celular/inmunología , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Citometría de Flujo , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Ligandos , Liposomas , Masculino , Neoplasias de la Próstata/patología , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Topotecan/administración & dosificación , Vinblastina/administración & dosificación , Vinblastina/análogos & derivados , VinorelbinaRESUMEN
We have used a naive human single-chain fragment variable (scFv) library as a source of random shape repertoire to directly probe the altered surface chemistry of tumor cells. We reported previously the identification of more than 90 internalizing phage monoclonal antibodies targeting prostate cancer cells, including those that are hormone refractory. In this report, we describe the conversion of a panel of those scFvs into full-length human immunoglobulins (IgGs) and show that tumor specificity is retained. We have further shown that antibodies isolated from a naive phage display library can nevertheless be of high affinity towards target tumor cells. In addition, full-length IgGs retain the functionality of parental scFvs including the ability to rapidly enter target cells through receptor-mediated endocytosis and thereby to mediate efficient and specific intracellular payload delivery to tumor cells. We have used recombinant IgGs to immunoprecipitate target antigens and analyzed their molecular composition by mass spectrometry. We have identified one target antigen as activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166 and have further studied tissue specificity of this internalizing ALCAM epitope by immunohistochemistry. Our study shows that cell type-specific internalizing human antibody can be readily identified from a naive phage antibody display library, characterized with regards to sequence, affinity, tissue specificity, and antigen identity, and modified genetically and chemically to generate various forms of targeted therapeutics.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Carcinoma/inmunología , Inmunoglobulina G/inmunología , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/inmunología , Molécula de Adhesión Celular del Leucocito Activado/inmunología , Molécula de Adhesión Celular del Leucocito Activado/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antineoplásicos/administración & dosificación , Células CHO , Carcinoma/patología , Carcinoma/terapia , Línea Celular Tumoral , Cricetinae , Cricetulus , Endocitosis/efectos de los fármacos , Humanos , Inmunoglobulina G/administración & dosificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Inmunohistoquímica , Inmunoprecipitación , Inmunoterapia/métodos , Liposomas , Masculino , Espectrometría de Masas , Biblioteca de Péptidos , Antígeno Prostático Específico/aislamiento & purificación , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunologíaRESUMEN
Diacylated (e.g. MALP-2) and triacylated (Pam(3)Cys derivatives) lipopeptides, deriving from the N-terminal moiety of respectively mycoplasmal and E. coli lipoproteins, are powerful adjuvants recognized by Toll-like receptors (TLR) which have been used successfully to trigger cell activation and immune responses. To design liposome-based vaccination constructs in which Th and CTL epitopes are conjugated to synthetic lipopeptide analogues anchored into the bilayers of the vesicles, the peptide moieties of the lipopeptides were functionalized with thiol-reactive groups, such as maleimide (Mal) or bromoacetyl, incorporated into liposomes and reacted with thiol carrying peptide epitopes. Because dendritic cells (DCs) play a key role as antigen-presenting cells in immune responses, in the present study we have evaluated the impact of the functionalization of lipopeptide analogues Pam(2)CAG, Pam(3)CAG and Ol(3)GAG on the phenotypic maturation of human monocyte-derived DCs. The intrinsic cellular activities of the lipopeptide analogues incorporated into liposomes were monitored, in vitro, by measuring the up-regulation of the cell-surface markers CD80, CD83, CD86 and HLA-DR. We found that in some cases their functionalization with thiol-reactive groups led to a loss of activity. The stimulatory potency can be ranked in the following order: Pam(3)CAG>/=Pam(2)CAG-Mal-Th approximately Pam(2)CAG-Mal>Pam(3)CAG-Mal-Th (where Th is a HS-peptide) and no appreciable activity was detected for Pam(3)CAG-Mal, Ol(3)CAG-Mal and Ol(3)CAG-Mal-Th. Our findings indicate that subtle modifications in the peptide moiety of lipopeptides have a great impact on the immunomodulatory properties of these molecules. For the engineering of liposome/lipopeptide-based vaccines, the maleimide derivative of Pam(2)CAG appears to be the best candidate.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Dendríticas/efectos de los fármacos , Lipoproteínas/farmacología , Liposomas/metabolismo , Adyuvantes Inmunológicos/biosíntesis , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/citología , Humanos , Lipoproteínas/biosíntesisRESUMEN
Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.