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Desenvolvimento e otimização de lipossomas encapsulados com citotóxicos de elevada potência para imunoterapia

Publication . Rico, Inês Casais e Costa; Silva, Frederico Nuno Castanheira Aires da; Gil, Solange Judite Roque Coelho Alves

O cancro é uma doença comum à Medicina Veterinária e Humana e é uma das principais causas de morte. Apesar das várias abordagens terapêuticas oncológicas existentes, a sua cura permanece como um desafio. No caso da quimioterapia, alguns dos motivos de insucesso prendem-se com: a toxicidade, a baixa eficácia e o desenvolvimento de resistências aos fármacos, apelando à investigação de novas abordagens terapêuticas que os superem. Os lipossomas, no âmbito da nanomedicina, têm sido investigados como transportadores de fármacos permitindo uma modulação da sua farmacocinética, do direcionamento para tecidos neoplásicos e redução dos efeitos secundários, apresentando, neste contexto, enorme potencial para a terapia oncológica. Deste modo, o presente estudo teve como objetivo o desenvolvimento e otimização de lipossomas para encapsulação de um fármaco citotóxico de elevada potência para imunoterapia. Para tal, foram avaliados os efeitos citotóxicos de sete iHADC – CI-994, panobinostat, SAHA, SBHA, scriptaid, trichostatin A e tubacina – tendo sido selecionado o panobinostat, com maior efeito citotóxico, para posterior encapsulação em lipossomas. Os efeitos citotóxicos do panobinostat lipossomal foram avaliados em linhas celulares de linfoma canino – CLBL-1 e 17-71 – e de glioblastoma humano – U87 – e comparados com os efeitos da doxorrubicina lipossomal. Foram ainda avaliados lipossomas associados a folatos como forma de direcionamento específico. Foram realizados três ensaios independentes para cada fármaco e em replicado para cada concentração. A viabilidade das células tratadas foi avaliada através do reagente alamarBlue® e os valores de IC50 calculados através de uma equação logarítmica no programa GraphPad Prism 6. Para análise estatística foi realizada uma ANOVA e teste de Tukey, através do programa R. Os resultados demonstraram atividade citotóxica dos iHDAC em CLBL-1 e 17-71, sendo capazes de induzir a acetilação de H3. Observou-se um maior efeito de citotoxicidade do panobinostat (IC50=20nM) que o da doxorrubicina (IC50=1050nM) com um valor de p<0,001. Os efeitos do panobinostat encapsulado (IC50=15nM) revelaram-se ser semelhantes ao do fármaco livre com um valor de p>0,05. Em U87, o panobinostat revelou ter também maior efeito citotóxico que a doxorrubicina. Os resultados obtidos sugerem que o panobinostat tem um grande efeito citotóxico em glioblastoma humano e linfoma canino. In vitro, o seu efeito citotóxico quando encapsulado em lipossomas sugeriu ser semelhante à formulação livre. Serão necessários estudos clínicos e translacionais para determinar a utilidade clínica e segurança do panobinostat lipossomal. No futuro, poderão ser acoplados anticorpos aos lipossomas para desenvolver um sistema de transporte de fármacos com grande potencial para Oncologia.

2019-01-17Master thesis

Open access

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Publication . Dias, Joana N. R.; Almeida, André; S. André, Ana; Aguiar, Sandra I; Bule, Pedro; Nogueira, Sara; Oliveira, Soraia S; Carrapiço, Belmira; Gil, Solange; Tavares, Luis; Aires Da Silva, Frederico

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.

2022-02-17Journal article

Open access

Clinical and immunological characterization of naturally occurring canine lymphoma : development and application of engineered recombinant antibodies for diagnosis and treatment

Publication . Dias, Joana Nunes Ribeiro; Tavares, Luís Manuel Morgado; Silva, Frederico Nuno Castanheira Aires da; Gil, Solange Judite Coelho Alves

Non-Hodgkin lymphoma (NHL) is one of the most common causes of cancer-related death worldwide. Although the outcome of NHL patients has improved with current therapies, the rate of mortality is still high. A plethora of new drugs is entering clinical development for NHL treatment; however, the approval of new treatments remains low due in part to the paucity of clinically relevant models for validation. Canine lymphoma (cNHL) shares remarkable similarities with its human counterpart, making the dog an excellent animal model to explore novel therapeutic options. Therefore, driven by the great success achieved by immunotherapies in human NHL, comparative research has focused on the development of similar immunotherapeutic approaches for dogs. However, the successful use of this animal model remains challenging, still lacking the characterization of the canine immune system, of common tumor epitopes, the development of canine-specific/cross-reactive agents and the establishment of preclinical models. Within this context, we aimed to develop novel antibody-based therapies for cNHL, while contributing for the characterization and validation of the cNHL model for translational immune-oncology research. For that purpose, a cNHL biobank was successfully constructed. The clinical cytokine patterns in patients with cNHL were investigated, confirming a local and systemic dysregulation in cytokine response. Furthermore, a positive correlation between intratumoral immune response and a favorable response to chemotherapy indicates that the modulation of the immune response might contribute to improve patient outcomes. With that in mind, a through characterization of canine CD20 expression was conducted and contributed for the validation of this receptor as a potential immunotherapeutic target for cNHL. This motivated the development and identification of a panel of single domain antibodies (sdAbs) with high binding activity and specificity to canine and human CD20. In addition, to develop a novel drug delivery system for cNHL treatment, we described novel methodologies to identify potential targets, while selecting highly specific sdAbs against NHL. This work allowed to select a promising pool of sdAbs that specifically target NHL tumor receptors for the development of a novel antibody drug conjugate (ADC). Furthermore, we conducted a thorough investigation of a novel ADC payload – panobinostat - a potent histone deacetylase (HDAC) inhibitor with strong in vitro and in vivo antitumor properties in cNHL. Finally, we established a new bioluminescent murine model for monitoring tumor progression and treatment response in preclinical studies. In summary, the work presented herein allowed the establishment of a solid platform for the acceleration of the translational research of novel immunotherapeutic approaches for comparative oncology.

2019-03-14Doctoral thesis

Open access