Development of novel antibody-drug conjugated molecules for treatment of B-cell malignancies

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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

Development of novel antibody-drug conjugated molecules for treatment of B-cell maligancies

Publication . André, Ana Filipa Santos; Silva, Frederico Nuno Castanheira Aires da; Tavares, Luís Manuel Morgado; Gonçalves, João Manuel Braz

Cancer is one of the leading causes of death worldwide, and is expected to continue increasing, with a projected 28.4 million cases by 2040. Dogs are also significantly affected by cancer, including NHL, which is one of the most common hematological malignancies in both species. Dogs are considered excellent models to accelerate the translation of treatments for human patients due to their similarities with humans. While immunotherapies, particularly monoclonal antibodies (mAbs), have brought specificity to cancer therapies, they are still mostly used in combination with conventional chemotherapy, which remains the standard of care for both species. However, conventional treatments still do not fully cure and have numerous adverse effects, highlighting the need for further improvement. To address this need, using the promising advantages of rabbit-derived sdAbs, we aimed to develop a platform for a novel sdAb drug delivery system for NHL treatment using the dog as an animal model. For this purpose, we explored three different drug delivery methods: ADCs, immunotoxins and immunoliposomes. For the development of ADC, we explored the potential of rabbit derived single-domain antibodies (sdAb) to selectively conjugate a payload towards cysteine at position 80. First, a rabbit-derived sdAb library against canine B-cell lymphoma receptors was subjected to in vitro and in vivo phage display. Then, VL sdAb that specifically targeted canine lymphoma cells in vitro and presented a good tumor uptake was selected for SN-38 site selective payload conjugation via its Cys80 and generated a stable and homogeneous C5- DAB-SN-38. This study validated a platform to develop novel ADCs that combine rabbit sdAbs benefits with the advantages of canine lymphoma model. Furthermore, this previously characterized and validated VL sdAb was also used to develop a new immunotoxin for the treatment of canine B-cell lymphoma. For that purpose, VL sdAb was conjugated with the PE38 toxin truncated form and tested in vitro in a canine B-cell lymphoma cells and in in vivo in a xenograft mouse model of canine lymphoma. This study validated immunotoxins as a potential treatment for canine lymphoma. Lastly, to validate a new liposome for canine lymphoma, we aimed to develop a liposome-based nanocarrier for panobinostat, using folate-targeted and non-targeted formulations. Both formulations were evaluated in canine lymphoma cells, validating liposomes as an effective treatment for canine lymphoma. In the future, our goal is to conjugate our VL sdAb to the liposome to obtain an immunoliposome for canine B-cell lymphoma. In conclusion, all the work developed contributed to the understanding of the importance of using the dog as a model and how these animals can contribute for clinical translation in the immune-oncology field

2023-11-03Doctoral 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

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

Publication . André, Ana; Dias, Joana N. R.; Aguiar, Sandra I; Nogueira, Sara; Bule, Pedro; Carvalho, Joana; António, João P. M.; Cavaco, Marco; Neves, Vera; Oliveira, Soraia; Vicente, Gonçalo; Carrapiço, Belmira; Braz, Berta São; Rütgen, Barbara; Gano, Lurdes; Correia, João D. G.; Castanho, Miguel A. R. B.; Gonçalves, João Rafael; Gois, Pedro M. P.; Gil, Solange; Tavares, Luis; Silva, Frederico Aires da

Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.

2023Journal article

Open access