RESUMEN
Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Monoclonales/administración & dosificación , Evaluación Preclínica de Medicamentos/métodos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Interleucina-23/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.
Asunto(s)
Anticuerpos/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Inmunoterapia/métodos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anciano , Anticuerpos/análisis , Anticuerpos/metabolismo , Femenino , Humanos , Intestinos/química , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/uso terapéutico , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: V565 is a novel oral anti-tumor necrosis factor (TNF)-α domain antibody being developed for topical treatment of inflammatory bowel disease (IBD) patients. Protein engineering rendered the molecule resistant to intestinal proteases. Here we investigate the formulation of V565 required to provide gastro-protection and enable optimal delivery to the lower intestinal tract in monkeys. METHODS: Enteric-coated V565 mini-tablets were prepared and dissolution characteristics tested in vitro. Oral dosing of monkeys with enteric-coated mini-tablets containing V565 and methylene blue dye enabled in vivo localization of mini-tablet dissolution. V565 distribution in luminal contents and feces was measured by enzyme-linked immunosorbent assay (ELISA). To mimic transit across the damaged intestinal epithelium seen in IBD patients an intravenous (i.v.) bolus of V565 was given to monkeys and pharmacokinetic parameters of V565 measured in serum and urine by ELISA. RESULTS: Enteric-coated mini-tablets resisted dissolution in 0.1 M HCl, before dissolving in a sustained release fashion at neutral pH. In orally dosed monkeys methylene blue intestinal staining indicated the jejunum and ileum as sites for mini-tablet dissolution. Measurements of V565 in monkey feces confirmed V565 survival through the intestinal tract. Systemic exposure after oral dosing was very low consistent with limited V565 mucosal penetration in healthy monkeys. The rapid clearance of V565 after i.v. dosing was consistent with renal excretion as the primary route for elimination of any V565 reaching the circulation. CONCLUSIONS: These results suggest that mini-tablets with a 24% Eudragit enteric coating are suitable for targeted release of orally delivered V565 in the intestine for topical treatment of IBD.
Asunto(s)
Anticuerpos/administración & dosificación , Antineoplásicos/administración & dosificación , Íleon/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/economía , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Anticuerpos/metabolismo , Antineoplásicos/farmacocinética , Química Farmacéutica/métodos , Heces , Concentración de Iones de Hidrógeno , Íleon/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Macaca fascicularis , Solubilidad , Comprimidos Recubiertos/administración & dosificación , Comprimidos Recubiertos/farmacocinéticaRESUMEN
TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.
Asunto(s)
Citocinas/sangre , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Infliximab , Mucosa Intestinal/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Biomarcadores/sangre , Colon/metabolismo , Colon/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Íleon/metabolismo , Íleon/patología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/patología , Infliximab/farmacocinética , Infliximab/farmacología , Mucosa Intestinal/patología , Masculino , Ratones , Factor de Necrosis Tumoral alfa/sangreRESUMEN
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.