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
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
Immunisation of BALB/c mice with a vaccine containing Vi polysaccharide conjugated to the Klebsiella pneumoniae outer membrane 40 kDa protein (rP40), in combination with Escherichia coli heat-labile toxin adjuvant (LT), elicited anti-Vi IgG antibodies after administration using different routes. Testing of the immune serum in opsonisation assays demonstrated the specific enhancement of Vi-positive bacterial uptake by cultured murine bone marrow derived macrophages. Intra-peritoneal challenge of mice immunised with the Vi-based vaccine elicited a degree of protection against virulent Vi+ Salmonella enterica serovar typhimurium (S. typhimurium). In contrast, Vi vaccination did not confer protection against oral challenge with virulent Vi-positive S. typhimurium or S. dublin.
Assuntos
Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Polissacarídeos Bacterianos/imunologia , Infecções por Salmonella/prevenção & controle , Animais , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Salmonella/imunologiaRESUMO
Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.