RESUMO
Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Doenças Autoimunes/terapia , Doenças do Complexo Imune/terapia , Fragmentos Fc das Imunoglobulinas/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Artrite/imunologia , Artrite/terapia , Artrite Experimental/imunologia , Artrite Experimental/terapia , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Linhagem Celular , Epidermólise Bolhosa Adquirida/imunologia , Epidermólise Bolhosa Adquirida/terapia , Humanos , Doenças do Complexo Imune/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Fagócitos , Ativação Plaquetária , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/terapia , Transdução de SinaisRESUMO
Unfractionated heparin is isolated from animal organs, predominantly porcine intestinal mucosa, and goes through an extensive process of purification before it can be used for pharmaceutical purposes. While the structural microheterogeneity of heparin is predominantly biosynthetically imprinted in the Golgi, subsequent steps involved in the purification and manufacture of commercial heparin can lead to the introduction of additional modifications. Postheparin crisis of 2008, it has become increasingly important to identify what additional structural diversity is introduced as a function of the purification process and thus can be determined as being heparin-related, as opposed to being an adulterant or contaminant, e.g., oversulfated chondroitin sulfate. Our study focuses on the identification of a previously unreported structure in heparin that arises due to specific steps used in the manufacturing process. This structure was initially observed as a disaccharide peak in a complete enzymatic digest of heparin, but its presence was later identified in the NMR spectra of intact heparin as well. Structural elucidation experiments involved isolation of this structure and analysis based on multidimensional NMR and liquid chromatography coupled with mass spectrometry (LC-MS). Heparin was also subjected to specific chemical reactions to determine which steps in the manufacturing process are responsible for this novel structure. Our results allowed for the definitive assignment of the structure of this novel process-related modification and enabled an identification of the putative steps in the process that give rise to the structure.
Assuntos
Dissacarídeos/química , Heparina/isolamento & purificação , Animais , Configuração de Carboidratos , Sulfatos de Condroitina/análise , Cromatografia Líquida , Glucuronidase/metabolismo , Heparina/química , Heparina Liase/metabolismo , Mucosa Intestinal/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução , Sulfatases/metabolismo , SuínosRESUMO
Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.
Assuntos
Progressão da Doença , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacologia , Melanoma Experimental/patologia , Mimetismo Molecular , Metástase Neoplásica , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Melanoma Experimental/irrigação sanguínea , Camundongos , Ressonância de Plasmônio de SuperfícieRESUMO
A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.