RESUMEN
BACKGROUND: Trastuzumab is a monoclonal antibody which demonstrates efficacy for HER2 positive breast cancer patients. Recently, an increased incidence of brain metastasis in trastuzumab-treated patients has been reported. The reason for this may be the effectiveness of systemic trastuzumab allowing patients to survive longer thus providing time for brain metastases to develop, along with the lack of penetration of systemic therapies through the blood brain barrier. In recent years, several administration routes to the brain have been evaluated. Albeit advances in the field, there is still a need for improved delivery of therapeutic antibodies to the brain. To address this challenge, we have developed two gene therapy-based methods enabling continuous secretion of active trastuzumab in the brain. METHODS: We have developed two gene therapy approaches for the delivery of the therapeutic anti-HER2 monoclonal antibody, trastuzumab, to the brain. We utilized the helper dependent adenovirus vector, containing trastuzumab light and heavy chains coding sequences (HDAd-trastuzumab). In the first approach, we used the Transduced Autologous Restorative Gene Therapy (TARGT) platform, in which dermal fibroblasts of human and mouse origin, are ex-vivo transduced with HDAd-trastuzumab vector, rendering continuous secretion of active trastuzumab from the cells locally. These genetically engineered cells were subsequently implanted intracranially to mice, contralateral to HER2 positive breast carcinoma cells inoculation site, enabling continuous secretion of trastuzumab in the brain. In the second approach, we used the same HDAd-trastuzumab viral vector, directly injected intracranially, contralateral to the HER2 positive breast carcinoma cells inoculation site. Both methods enabled therapeutic concentrations of local in-vivo production of active trastuzumab in a mouse model of brain metastatic breast cancer. RESULTS: Trastuzumab secreted from the TARGT platform demonstrated in-vitro affinity and immune recruitment activity (ADCC) similar to recombinant trastuzumab (Herceptin, Genentech). When implanted in the brain of HER2 positive tumor-bearing mice, both the TARGT platform of dermal fibroblasts engineered to secrete trastuzumab and direct injection of HDAd-trastuzumab demonstrated remarkable intracranial tumor growth inhibitory effect. CONCLUSIONS: This work presents two gene therapy approaches for the administration of therapeutic antibodies to the brain. The TARGT platform of dermal fibroblasts engineered to secrete active trastuzumab, and the direct injection of HDAd-trastuzumab viral vector, both rendered continuous in-vivo secretion of active trastuzumab in the brain and demonstrated high efficacy. These two approaches present a proof of concept for promising gene therapy based administration methods for intracranial tumors as well as other brain diseases.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/terapia , Neoplasias de la Mama/patología , Técnicas de Transferencia de Gen , Trastuzumab/uso terapéutico , Adenoviridae/genética , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/metabolismo , Neoplasias Encefálicas/patología , Neoplasias de la Mama/terapia , Células Cultivadas , Sistemas de Liberación de Medicamentos/métodos , Femenino , Fibroblastos/metabolismo , Fibroblastos/trasplante , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Transducción Genética , Trastuzumab/administración & dosificación , Trastuzumab/genéticaRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is a symmetric inflammatory polyarthritis associated with high concentrations of pro-inflammatory, cytokines including tumor necrosis factor (TNF)-α. Adalimumab is a monoclonal antibody (mAb) that binds TNF-α, and is widely used to treat RA. Despite its proven clinical efficacy, adalimumab and other therapeutic mAbs have disadvantages, including the requirement for repeated bolus injections and the appearance of treatment limiting anti-drug antibodies. To address these issues, we have developed an innovative ex vivo gene therapy approach, termed transduced autologous restorative gene therapy (TARGT), to produce and secrete adalimumab for the treatment of RA. METHODS: Helper-dependent (HD) adenovirus vector containing adalimumab light and heavy chain coding sequences was used to transduce microdermal tissues and cells of human and mouse origin ex vivo, rendering sustained secretion of active adalimumab. The genetically engineered tissues were subsequently implanted in a mouse model of RA. RESULTS: Transduced human microdermal tissues implanted in SCID mice demonstrated 49 days of secretion of active adalimumab in the blood, at levels of tens of microgram per milliliter. In addition, transduced autologous dermal cells were implanted in the RA mouse model and demonstrated statistically significant amelioration in RA symptoms compared to naïve cell implantation and were similar to recombinant adalimumab bolus injections. CONCLUSIONS: The results of the present study report microdermal tissues engineered to secrete active adalimumab as a proof of concept for sustained secretion of antibody from the novel ex vivo gene therapy TARGT platform. This technology may now be applied to a range of antibodies for the therapy of other diseases.
Asunto(s)
Adalimumab/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de Enfermedad , Factor de Necrosis Tumoral alfa/metabolismo , Adalimumab/farmacocinética , Animales , Anticuerpos Monoclonales/farmacocinética , Citocinas/metabolismo , Femenino , Ingeniería Genética , Terapia Genética , Humanos , Masculino , Metotrexato/farmacología , Ratones , Ratones SCID , Resultado del TratamientoRESUMEN
Infectious bronchitis virus (IBV) is prevented primarily by the use of live attenuated vaccines, which are known to have a limited strain range of protection. Alternative vaccines against the emerging new virus strains can improve control of the disease. The aim of this study was to evaluate the immunogenic potential of two recombinant viral proteins, when administered by eyedrop, without the assistance of a vector. The recombinant S1 (rS1) and N (rN) proteins of the M41 strain expressed in E. coli were tested, and the live attenuated vaccine H120 was used as a positive control. Protection was evaluated by re-isolation of virus from tracheas of vaccinated chickens after challenge with strain M41. After three immunizations, rS1 glycoprotein induced 40% protection, while vaccination with rN provided no protection. Vaccination with rS1, rN, or H120 induced a cellular immune response as demonstrated by in vitro ChIFN-γ production by splenocytes of vaccinated birds. Vaccination with H120, and to a lesser extent rS1, induced HI and virus-specific IgG antibody production. These findings indicate that recombinant viral proteins administered through the mucosal route can evoke an immune response without the assistance of a vector.
Asunto(s)
Virus de la Bronquitis Infecciosa/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Enfermedades de las Aves de Corral/inmunología , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Administración a través de la Mucosa , Animales , Anticuerpos Antivirales/sangre , Pollos , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Proteínas de la Nucleocápside de Coronavirus , Virus de la Bronquitis Infecciosa/metabolismo , Interferón gamma/biosíntesis , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/genética , Proteínas de la Nucleocápside/administración & dosificación , Proteínas de la Nucleocápside/genética , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Glicoproteína de la Espiga del Coronavirus , Vacunación , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Passive immunization with cross-species antibodies triggers the patient's immune response, thereby preventing repeated treatment. Mannosamine-biotin adduct (MBA) has been described as a masking agent for immunogenic reduction and here, the immunogenicity and biological activity of MBA-coated horse anti-viper venom (hsIgG) were compared to those of uncoated or PEGylated hsIgG. In in vitro tests, hsIgG binding was not affected by MBA conjugation. The immune response to hsIgG-MBA was about 8-fold and 32-fold lower than to PEG-coated and uncoated hsIgG, respectively. In vivo, hsIgG-MBA showed efficient venom-neutralization activity. We thus demonstrate the feasibility of using MBA as a masking agent for passive immunization with cross-species antibodies.
Asunto(s)
Antivenenos/química , Antivenenos/inmunología , Biotina/metabolismo , Reacciones Cruzadas , Hexosaminas/metabolismo , Inmunización Pasiva/métodos , Venenos de Serpiente/antagonistas & inhibidores , Animales , Caballos , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Neutralización , Unión Proteica , Venenos de Serpiente/inmunologíaRESUMEN
A variety of protein-coating procedures are used to modify proteins' properties. The principle coating agent used is PEGylation, in which proteins are coated by conjunction to polyethylene glycol (PEG). In the present study, we describe a novel approach that makes use of small molecules with multifunctional groups as the protein-coating agent. The new coating molecule was produced by reacting two endogenous molecules, mannosamine and biotin, to form mannose-biotin adducts (MBA). hIgG was coated with MBA at various MBA/protein ratios. The immunogenicity of MBA-coated hIgG was tested in chickens. A dose-responsive effect of MBA/hIgG ratio on immune response suppression was detected, with an optimal masking effect at a 12:1 ratio. The immune response to MBA-coated hIgG was about eightfold lower than that to PEG-coated hIgG. MBA also increased antibody-antigen-binding affinity, and decreased recognition of the Fc domain of MBA-coated hIgG by Fc receptor and secondary antibodies. While the PEG molecule consists of inert repeating units of ethylene oxide with no additional functional group to allow for potentially desirable modifications, the MBA has several functional groups, including vicinal hydroxyls, which can easily be converted to active residues such as aldehydes or carboxyls. This may be of importance for developing passive immunizations or for achieving tolerance of the immune response to an immunogenic molecule or virus. In summary, we developed a new protein-coating molecule with the ability to mask foreign antigens and in the case of antibodies, to enhance activity.