RESUMO
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.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-23/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Anticorpos/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Imunoterapia/métodos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Anticorpos/análise , Anticorpos/metabolismo , Feminino , Humanos , Intestinos/química , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
Assuntos
Anticorpos/administração & dosagem , Antineoplásicos/administração & dosagem , Íleo/efeitos dos fármacos , Doenças Inflamatórias Intestinais/economia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Anticorpos/metabolismo , Antineoplásicos/farmacocinética , Química Farmacêutica/métodos , Fezes , Concentração de Íons de Hidrogênio , Íleo/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Macaca fascicularis , Solubilidade , Comprimidos com Revestimento Entérico/administração & dosagem , Comprimidos com Revestimento Entérico/farmacocinéticaRESUMO
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.
Assuntos
Citocinas/sangue , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab , Mucosa Intestinal/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Biomarcadores/sangue , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Íleo/metabolismo , Íleo/patologia , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Infliximab/farmacocinética , Infliximab/farmacologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Fator de Necrose Tumoral alfa/sangueRESUMO
In Gram-negative bacteria, quorum sensing control of gene expression is mediated by transcription factors of the LuxR family, whose DNA-binding affinity is modulated by diffusible N-acyl homoserine lactone (AHL) signalling molecules. In Serratia sp. ATCC 39006 and the plant pathogen Erwinia carotovora ssp. carotovora (Ecc), the biosynthesis of the ß-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (Car) is under quorum sensing control. This study has revealed that, uniquely, the LuxR family transcriptional activator CarR(39006) from Serratia 39006 has no detectable affinity for cognate AHL molecules. Furthermore, CarR(39006) was shown to be naturally competent to bind to its target promoter with high affinity, activate transcription and resist cellular proteolysis, and was unaffected by AHL signals. Experiments with chimeric proteins suggest that the C-terminal DNA-binding domain of CarR(39006) may be responsible for conferring AHL independence. In contrast, we show that the homologous CarR(Ecc) protein binds to its 3O-C6-HSL ligand with high affinity, and that the highly conserved Trp-44 residue is critical for this interaction. Unlike TraR from Agrobacterium tumefaciens, CarR(Ecc) is not directly protected from cellular proteolysis by AHL binding, but via AHL-induced DNA binding. At physiological protein concentrations, AHL binding induces CarR(Ecc) to bind to its target promoter with higher affinity and activate transcription.
Assuntos
Percepção de Quorum , Proteínas Repressoras/metabolismo , Serratia/genética , Transativadores/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Serratia/metabolismo , Transdução de Sinais/genética , Transativadores/genéticaRESUMO
Many Gram-negative bacteria use quorum sensing (QS) to regulate expression of multiple genes, by utilizing small diffusible signalling molecules called N-acyl homoserine lactones (acyl-HSLs). Serratia sp. ATCC 39006 produces the red pigment prodigiosin under QS control, in response to the short-chain signal C4-HSL. In this study, we have demonstrated that an acyl-HSL-deficient mutant can be used as a visual biosensor to detect short-chain acyl-HSLs. We have quantified the acyl-HSL sensitivity spectrum of the Serratia 39006 prodigiosin QS system, and have demonstrated a strong specificity for the natural ligand C4-HSL. Mutations in the pigX and pigZ genes in Serratia 39006 resulted in an overproduction of prodigiosin, caused by increased transcription of the prodigiosin biosynthetic operon. A new biosensor (SP19) with enhanced prodigiosin production was created by addition of pigX and pigZ mutations to the existing biosensor. We have demonstrated that SP19 is superior to biosensor strains CV026 and Agrobacterium NTL4 (pZLR4) for the detection of short-chain acyl-HSLs present in spent culture supernatants. Researchers working with QS bacteria that produce short-chain acyl-HSLs can use strain SP19 as a simple visual acyl-HSL biosensor with no requirement for expensive detection equipment.
RESUMO
The ability to utilize the siderophore ferrichrome as an iron source was found to be a variable trait in a field population of mesorhizobia. To investigate the genetic basis of this variation, genes required for ferrichrome utilization (fhu genes) were characterized in Mesorhizobium strain R88B, an Fhu(+) member of the population. Functional fhu genes were present at three loci. Two genes of the ferrichrome ABC transporter, fhuBD, were identified at an fhu1 locus downstream of the symbiosis island that was integrated at the phe-tRNA gene. The fhuA gene encoding the ferrichrome outer membrane receptor was located in the fhu2 locus together with non-functional fhuDB genes, while the fhuC gene encoding the ATPase required for ferrichrome transport was part of the fhu3 locus that included genes required to form a functional TonB complex. None of the fhu genes were present in the sequenced Mesorhizobium loti strain MAFF303099. Comparisons with MAFF303099 suggested that the fhu2 and fhu3 loci evolved through small-scale (< 5 kb) acquisitions and deletions. Despite their independent origins, the three fhu loci were coordinately regulated in response to iron availability. Within the mesorhizobial population, the ability to utilize ferrichrome was most strongly correlated with the presence of the fhuA gene. We hypothesize that the ferrichrome transport system evolved through cycles of gene acquisition and deletion, with the positive selection pressure of an iron-poor or siderophore-rich environment being offset by the negative pressure of the outer membrane receptor being a target for phage.