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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2022.tde-08082022-094847
Document
Author
Full name
Renata Nacasaki Silvestre
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2022
Supervisor
Committee
Castro, Virgínia Picanço e (President)
Figueiredo, Marxa Leão
Santis, Gil Cunha de
Silva, Thiago Aparecido da
Title in Portuguese
Geração de células NK-CAR anti-CD19 como uma alternativa alogênica para o tratamento de neoplasias de células B
Keywords in Portuguese
Células natural killer
Imunoterapia
Modificações genéticas
NK-CAR
Receptor quimérico de antígeno
Abstract in Portuguese
Recentemente, a terapia de neoplasias hematológicas foi revolucionada pelo uso de células T autólogas que expressam receptores quiméricos de antígenos (CAR). Apesar desse sucesso, existem inúmeras vantagens para explorar também células CAR alogênicas, como as CAR-Natural Killer (NK-CAR). A capacidade de direcionar a citotoxicidade contra tumores refratários por meio da expressão de CAR em NK provavelmente contribuirá para uma mudança de paradigma no tratamento do câncer. Nossa hipótese é que novos construtos CAR que modulam as vias de IL-15 ou IL-27 podem promover aumentos significativos na proliferação, ativação e atividade citotóxica de células NK. Células NK de diferentes fontes (linhagem NK-92, sangue periférico e de cordão) foram modificadas geneticamente e analisadas por citometria de fluxo, por meio da marcação da região fab´ de CAR. As células NK-92 transduzidas foram selecionadas positivamente por coluna magnética. Células NK-CAR tiveram sua citotoxicidade avaliada por três metodologias: citometria de fluxo, Delfia Europium e Incucyte System. A eficiência in vivo das células NK-CAR foi avaliada em modelos murinos de neoplasia de células B, pela medição da carga tumoral por bioluminescência e curva de sobrevida. Inicialmente, exploramos o potencial dos promotores EF1α e SFFV para conduzir a expressão de CAR. O promotor SFFV produziu altos títulos lentivirais e alcançou a melhor eficácia de transdução em células NK. No vetor SEW-SFFV-CAR.19, clonamos as citocinas IL-15, IL-15/IL-15Rα e IL-27. Células NK de diferentes fontes foram então modificadas com estes vetores e a porcentagem de NK-CAR foi mensurada por citometria de fluxo. Nossos resultados demonstraram uma baixa porcentagem de células expressando SEW-SFFV-CAR.19 (10,7%±1,93), SEW-SFFV-CAR.19-IL-15 (5,8%±3), SEW-SFFV-CAR.19-IL-15/IL-15Rα (3,7%±1,1) e SEW-SFFV-CAR.19-IL-27 (3,2%±0,6) em células NK-92. As células NK-92-CAR foram selecionadas com anticorpo anti-CAR, o que proporcionou um aumento na pureza de células NK-CAR (>98% CAR+). Posteriormente, células NK-CAR foram co-cultivadas com células tumorais que expressavam CD19 ou não. O ensaio de citotoxicidade mostrou que células NK-92-CAR.19 (todos os constructos) foram mais citotóxicas contra células CD19+ quando comparadas com NK-92 wt (p<0,05). Células NK de sangue periférico (NK-PB) e de sangue de cordão (NK-CB) apresentaram taxas de eficiência média de transdução de 26,72% e 54,5%, respectivamente, entretanto essa expressão não foi estável. Apesar disso, ensaios citotóxicos demonstraram que células NK-PB-SFFV-CAR.19 e NK-CB-SFFV-CAR.19 foram mais citotóxicas contra células tumorais que expressam CD19 do que células NK não transduzidas (p<0,05). Na avaliação da eficiência antitumoral das células NK-92-CAR.19 in vivo não houve diminuição na carga tumoral entre os grupos controle e tratamentos até dia 14, porém no dia 21 notou-se uma menor carga tumoral no grupo tratado com células NK-92-SFFV-CAR.19-IL-15/IL-15Rα (p<0,05), o qual também apresentou uma maior sobrevida (p<0,05). Portanto, nossos dados demonstram que é viável gerar células NK modificadas por CAR com atividade antitumoral potente e seletiva, mas ajustes no modelo animal ainda são necessários. Em resumo, esse projeto de pesquisa deu o passo inicial para a implementação da geração e produção de NK-CAR no Brasil. Os resultados deste projeto representam o início do desenvolvimento de uma plataforma off-the-shelf de células NK-CAR que poderá, num futuro próximo, ser utilizada na imunoterapia alogênica do câncer.
Title in English
Generation of anti-CD19 CAR-NK cells as an allogeneic alternative for B-cell neoplasia treatment
Keywords in English
CAR-NK
Chimeric antigen receptor
Genetic modifications
Immunotherapy
Natural Killer cells
Abstract in English
Recently, hematologic malignancy therapy has been upgraded by the use of autologous T cells that express chimeric antigen receptors (CAR). Despite this success, there are many advantages to investing as well in allogeneic CAR, for example CAR-natural killer cells (CAR-NK). The ability to target powerfully cytotoxicity against refractory tumors through CAR expression in NK is likely to contribute to a paradigm shift in cancer treatment. Our hypothesis is that new CAR constructs that modulate IL-15 or IL-27 pathways can promote significant increases in NK cell proliferation, activation, and cytotoxic activity. NK cells from different sources (NK-92 cell line, peripheral blood and cord blood) were genetically modified and analyzed by flow cytometry, through labeling the Fab´ region of the CAR receptor. The transduced NK-92 cells were positively selected by magnetic column. CAR-NK cells had their cytotoxicity evaluated by three methodologies: flow cytometry, Delfia Europium and the Incucyte System. In vivo CAR-NK efficiency was evaluated in a murine model of B-cell neoplasia. Tumor burden was measured by bioluminescence and the curve of survival was also analyzed. Initially, we explored the potential of EF1α and SFFV promoters to establish CAR expression. SFFV promoter produced high lentiviral titers and achieved the best transduction efficiency in NK cells. We cloned IL-15, IL-15/IL- 15Rα and IL-27 cytokines in the SEW-SFFV-CAR.19 vector. NK cells from different sources were then modified with these vectors and CAR-NK percentage were measured by flow cytometry. Our results demonstrated a low percentage of cells expressing SEW-SFFV-CAR.19 (10,7%±1,93), SEW-SFFV-CAR.19-IL-15, (5,8%±3), SEW-SFFV-CAR.19IL-15/IL-15Rα (3,7%±1,1) and SEW-SFFV-CAR.19-IL-27 (3,2%±0,6) in NK-92 cells. CAR.19-NK-92 cells were selected with anti-CAR antibody, which caused an enrichment in CAR-NK cells (>98% CAR+). Subsequently, CAR-NK cells were co-cultured with tumor cells expressing CD19 or not. Cytotoxicity assay showed that CAR-NK-92 (all constructs) were more cytotoxic against CD19+ cells when compared with NK-92 wt (p<0.05). Peripheral blood NK cells (NK-PB) and cord blood NK cells (NK-CB) showed transduction efficiency rates of 26,72% and 54,5%, respectively, however this expression was not stable. Nevertheless, cytotoxic assays showed that NK-PB-SFFV-CAR.19 and NK-CB-SFFV-CAR.19 cells were more cytotoxic against tumor cells expressing CD19 than non-transduced NK cells (p<0.05). In the evaluation of CAR.19-NK-92 cells antitumoral efficacy in vivo, there was no significant decrease in tumor burden between the control and treatment groups until day 14, but on day 21 there was a decrease in tumor burden in the group treated with NK-92-SFFV-CAR.19-IL-15/IL-15Rα (p<0.05), which also showed a longer survival rate (p<0.05). In conclusion, our data showed that it is possible to generate CAR modified NK cells with a potent and selective antitumoral activity, but some readjustments in the animal model are required. In summary, this research project took the initial step towards the generation and production of CAR-NK in Brazil. The results of this project represent the beginning of the development of a CAR-NK cells off-theshelf platform, which might soon be used in allogeneic cancer immunotherapy.
 
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Publishing Date
2022-08-16
 
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