RESUMO
Anti-idiotypic antibodies (anti-Id) as surrogate TAAs have been shown to induce immunity against tumours in animals and humans. SM262 is a human monoclonal anti-Id raised against MAb 17-1A recognising Ep-CAM. Plasmids encoding the variable regions of SM262 with either murine or human Fc regions, both with and without fusion to GM-CSF were constructed. DNA was delivered by gene gun to C57BL/6 (wt) mice and mice expressing the transgene for human Ep-CAM (tg). The immunogenicity of anti-Id DNA constructs, anti-Id protein and Ep-CAM DNA vaccines was compared. SM262 plasmids induced antibodies (Abs) inhibiting MAb 17-1A binding to SM262 as well as recognising Ep-CAM in wt and tg mice. Fusion to GM-CSF evoked significantly higher Ab titres, whereas a xenogeneic Fc region had no significant effect. The highest Ab titres were elicited by protein immunisation. The original Ag was superior as compared to the anti-Id vaccines in wt but not tg mice in terms of Ab induction. A weak Ep-CAM-specific cytotoxic response was induced in wt but not tg mice. The data suggest that B cell tolerance to Ep-CAM can be circumvented by anti-Id DNA, anti-Id protein as well as Ep-CAM DNA immunisation. Fusion of GM-CSF to anti-Id increased the magnitude of the immune response with no requirement of a foreign Fc domain. Furthermore, no superiority of Ep-CAM as compared to anti-Id DNA vaccine was noted in tg mice and protein immunisation induced a more potent humoral response than DNA. The results might have implications for the design of future vaccine trials using Ep-CAM as a target structure.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Vacinas Anticâncer/imunologia , Animais , Antígenos de Neoplasias , Biomarcadores Tumorais , Complexo CD3 , Moléculas de Adesão Celular , Molécula de Adesão da Célula Epitelial , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PlasmídeosRESUMO
The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) 'clamps' for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by 'hybrid' phosphorothioate-LNA ODNs induces tumour necrosis factor-alpha production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.
Assuntos
Oligonucleotídeos/química , Plasmídeos/química , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Ilhas de CpG/genética , Cricetinae , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Corantes Fluorescentes/química , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Oligonucleotídeos/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
BACKGROUND: IL-16 is an important T-cell chemotactic cytokine in asthmatic airways; its release from allergen-stimulated bronchial mucosa in mild asthma has been shown to be dependent on CD28/B7 costimulation. OBJECTIVE: We have extended our previous studies to investigate the role of IL-16 and CD28/B7 costimulation in T-lymphocyte chemotactic activity (TLCA) released from the bronchial mucosa in more severe asthma. METHODS: TLCA was determined in the supernatants of induced sputum and allergen-stimulated bronchial mucosal explants from healthy volunteers and volunteers with mild and moderately severe asthma by means of a Boyden chamber technique. The contribution of IL-16 to the activity was evaluated through use of a neutralizing monoclonal antibody; the contribution of CD28/B7 costimulation to allergen-induced release of TLCA was determined through use of CTLA4-Ig fusion protein and neutralizing monoclonal antibodies to CD80 (B7.1) and CD86 (B7.2). RESULTS: Induced sputum and unstimulated explants from asthmatic subjects generated significant spontaneous TLCA (P <.05). Both mild and moderate asthmatic explants showed significantly elevated Dermatophagoides pteronyssinus -induced release of TLCA, but only in mild asthma could sputum and allergen-stimulated explant TLCA be inhibited by anti-IL-16 (median inhibition, 39% and 59%; P <.05). In addition, allergen released significant quantities of IL-16 from mild asthmatic explants (P <.05) but not from moderate asthmatic explants. Antibodies to the CD28 counter-ligands CD80 and CD86 inhibited allergen-induced release of TLCA in mild asthmatic explants by 94% (P <.05) and 62%, but TLCA release from moderate asthmatic explants was unaffected by CTLA4-Ig. CONCLUSION: These results show that TLCA release in moderate asthmatic airways, in contrast to mild asthmatic airways, is not dependent on CD28/B7 costimulation and does not involve IL-16.