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Combination of a multifunctional nanovaccine with modulators of pancreatic cancer microenvironment
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Resumo(s)
O adenocarcinoma ductal do pâncreas (ADP) é o subtipo de cancro pancreático mais prevalente e agressivo com uma alarmante taxa de sobrevivência a cinco anos de apenas 9%. A imunoterapia tem surgindo como uma modalidade de tratamento promissora para o tratamento deste tipo de tumor, focando-se na utilização de vacinas anti tumorais e inibidores dos immune checkpoints. No entanto, estas abordagens imunoterapêuticas não têm trazido benefícios para doentes com ADP devido à imunossupressão marcada, aliada a um microambiente tumoral altamente fibrótico e também a respostas imunitárias pouco eficazes, características deste tipo de cancro. Deste modo, novas estratégias imunoterapêuticas estão a ser desenvolvidas para ultrapassar estes desafios, passando na sua maioria por combinações de abordagens terapêuticas com mecanismos de ação complementares. Este trabalho baseia-se na hipótese de que uma vacina à base de nanopartículas multifuncionais possa ter um efeito sinérgico quando combinada com um anticorpo monoclonal que ative o recetor 4-1BB e/ou um inibidor da via de sinalização FAK. Esta combinação promoverá respostas citotóxicas resultantes da ativação e expansão de linfócitos T. Para testar esta hipótese, começámos por desenvolver e caracterizar as propriedades físico-químicas de uma Nanovacina constituída por ácido polilático (PLA) e ácido polilático co-glicólico (PLGA) conjugado com manose. Esta formulação foi desenvolvida no sentido de permitir a incorporação de sequências longas e curtas de péptidos específicos de proteínas expressas no ADP, bem como agonistas de recetores Toll-like. Pretende-se que a Nanovacina direcione as células dendríticas (DC) para os nódulos linfáticos e assim promova a sua ativação e maturação, o que levará à estimulação da resposta imunitária adaptativa. As nanopartículas apresentaram um diâmetro médio entre os 200 e os 220 nm, um índice de polidispersão (PdI) baixo, um potencial zeta próximo da neutralidade e uma eficiência de encapsulação (EE) superior a 60%. Tanto as nanopartículas formuladas sem manose (NP) como as formuladas com manose (man-NP) não afetaram a viabilidade in vitro das linhas celulares tumorais KPC e Panc02, bem como da linha celular imatura de DC, JAW SII. Adicionalmente, as man-NP foram maioritariamente internalizadas pelas DC migratórias, induzindo a ativação e maturação destas DC in vivo, o que foi evidenciado pelo aumento marcado da expressão dos marcadores de superfície CD80 e CD86. Foi desenvolvido um modelo animal de ADP tendo por base a inoculação subcutânea de células KPC em ratinho. Os ratinhos com tumor foram tratados com a Nanovacina combinada com agonistas dos immune checkpoints e moduladores do microambiente tumoral do ADP. O crescimento tumoral foi semelhante nos grupos tratados. No entanto, observou-se a estimulação das células NK T, bem como a ativação de DC CD8+ citotóxicas, no baço e tumores de animais tratados com a combinação tripla. Estes resultados demonstram que existiu um efeito sinérgico entre a Nanovacina, agonistas do 4-1BB e o modulador da via associada à imuno evasão deste tumor. Deste modo, esta Nanovacina apresenta o potencial de regular a resposta imunológica local nestes tumores, permitindo a sua sensibilização a outras imunoterapias.
Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive subtype of pancreatic cancer with a dismal 5-year overall survival of only 9%. Immunotherapy, focused on the use of cancer vaccines and immune checkpoint inhibitors, has been emerging as a promising alternative against these unmet medical needs. Nonetheless, it has failed to provide clinical benefits in PDAC, due to limited specific immune responses mostly resultant from the marked immunosuppression and desmoplastic tumor microenvironment characteristic of this type of cancer. Novel combinational immunotherapeutic strategies are being developed to overcome these challenges currently reported for PDAC treatment. We hypothesized that a multifunctional nanoparticulate cancer vaccine may have a synergistic effect, when combined with α4-1BB and/or a FAK inhibitor (Defactinib) to promote effective cytotoxic antitumor immune responses, mediated by T-cell stimulation and expansion. For this purpose, a mannose-grafted poly(lactic-co-glycolic) acid and poly(lactic) acid (PLGA/PLA) Nanovaccine, with entrapped PDAC-associated MHCI or MHCII peptides and Toll-like receptors immune potentiators, was developed to target dendritic cells (DC) on lymph nodes, promote their activation and maturation and, consequently prime T-cell immune responses. Formulated nanoparticles presented a mean size diameter between 200-220 nm, a low polydispersity index (PdI), a surface charge close to neutrality and an acceptable entrapment efficiency (EE) superior to 60%. Both non-mannosylated (NP) and mannosylated (man-NP) nanoparticles did not affect the in vitro cell viability of the tumor cell lines KPC and Panc02, and the immature DC cell line, JAW SII. In addition, man-NP were highly internalized by migratory DC, and induced DC activation and maturation, which was shown by the increased expression of the surface markers CD80 and CD86 in vivo. A KPC xenograft mouse model of PDAC was developed and tumor-bearing mice were treated with the Nanovaccine combined with immune checkpoint agonists and modulators of PDAC tumor microenvironment. Tumor growth was similar among the treatment groups, however FACS analysis of spleen and tumor collected from nanovaccine-treated mice evidenced an increase in the systemic cytotoxic immune responses with a marked stimulation of NK T cells. Moreover, a significant activation of conventional DC that present antigens to naïve CD8+ cytotoxic T cells, where noted in this treatment group. These findings support the synergism between polymeric mannosylated nanoparticles, as promising drug delivery systems for cancer vaccination, in combination with other immunotherapeutic agents such as costimulatory agonists and modulators of PDAC-related pathways, providing important information for a future establishment of a novel combination therapy against PDAC.
Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive subtype of pancreatic cancer with a dismal 5-year overall survival of only 9%. Immunotherapy, focused on the use of cancer vaccines and immune checkpoint inhibitors, has been emerging as a promising alternative against these unmet medical needs. Nonetheless, it has failed to provide clinical benefits in PDAC, due to limited specific immune responses mostly resultant from the marked immunosuppression and desmoplastic tumor microenvironment characteristic of this type of cancer. Novel combinational immunotherapeutic strategies are being developed to overcome these challenges currently reported for PDAC treatment. We hypothesized that a multifunctional nanoparticulate cancer vaccine may have a synergistic effect, when combined with α4-1BB and/or a FAK inhibitor (Defactinib) to promote effective cytotoxic antitumor immune responses, mediated by T-cell stimulation and expansion. For this purpose, a mannose-grafted poly(lactic-co-glycolic) acid and poly(lactic) acid (PLGA/PLA) Nanovaccine, with entrapped PDAC-associated MHCI or MHCII peptides and Toll-like receptors immune potentiators, was developed to target dendritic cells (DC) on lymph nodes, promote their activation and maturation and, consequently prime T-cell immune responses. Formulated nanoparticles presented a mean size diameter between 200-220 nm, a low polydispersity index (PdI), a surface charge close to neutrality and an acceptable entrapment efficiency (EE) superior to 60%. Both non-mannosylated (NP) and mannosylated (man-NP) nanoparticles did not affect the in vitro cell viability of the tumor cell lines KPC and Panc02, and the immature DC cell line, JAW SII. In addition, man-NP were highly internalized by migratory DC, and induced DC activation and maturation, which was shown by the increased expression of the surface markers CD80 and CD86 in vivo. A KPC xenograft mouse model of PDAC was developed and tumor-bearing mice were treated with the Nanovaccine combined with immune checkpoint agonists and modulators of PDAC tumor microenvironment. Tumor growth was similar among the treatment groups, however FACS analysis of spleen and tumor collected from nanovaccine-treated mice evidenced an increase in the systemic cytotoxic immune responses with a marked stimulation of NK T cells. Moreover, a significant activation of conventional DC that present antigens to naïve CD8+ cytotoxic T cells, where noted in this treatment group. These findings support the synergism between polymeric mannosylated nanoparticles, as promising drug delivery systems for cancer vaccination, in combination with other immunotherapeutic agents such as costimulatory agonists and modulators of PDAC-related pathways, providing important information for a future establishment of a novel combination therapy against PDAC.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Polymeric nanovaccine Pancreatic ductal adenocarcinoma Tumor microenvironment PDAC-related signaling pathways Teses de mestrado - 2021