Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo

IntroductionNatural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary.MethodsIn this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence.ResultsWe demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model.ConclusionTogether with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals

Production of lentiviral vectors under scalable conditions for the generation of CAR-T cells for cell therapy

Os vetores lentivirais são muito utilizados como vetores para a modificação gênica em terapias celular e gênica como as que empregam células T-CAR, devido a sua estabilidade de integração no genoma das células, eficiência de transdução e segurança. A produção destes vetores em cultivos em monocamada em meios contendo soro fetal bovino (SFB), tradicionalmente empregada na academia e centros de pesquisa, possui uma série de limitações, dentre elas a incompatibilidade com uma produção de forma mais controlada em maiores escalas, sendo desejável para isto o cultivo celular em suspensão em biorreatores. O objetivo deste projeto foi produzir partículas lentivirais em condições escalonáveis, utilizando células adaptadas para crescimento em suspensão em meio de cultura livre de soro fetal bovino (SFB). Portanto, células da linhagem HEK293T, adaptadas ao cultivo em suspensão, foram utilizadas para produção de partículas lentivirais que expressam a proteína sintética CAR anti-CD19 assim como IL-18 e GFP. A primeira etapa consistiu em produzir partículas lentivirais em monocamada em meio contendo SFB como condição controle e avaliar sua funcionalidade. Em monocamada a produção média, em 48 horas, foi de 8x106 partículas/mL. A funcionalidade do vetor foi avaliada por meio da modificação de linfócitos T para expressão da molécula CAR. Os resultados indicaram que o vetor é capaz de induzir resposta antitumoral nos linfócitos T, que por sua vez foram capazes de identificar e levar a morte células CD19 positivas in vitro. As células HEK293T adaptadas tiveram seu perfil cinético e metabólico caracterizado, tendo sido obtidos resultados satisfatórios com e sem suplementação com Cellboost. A presença deste componente, no entanto, inibiu a transfecção transiente e por conseguinte a produção de partículas lentivirais. Mesmo na ausência de Cellboost, a produção lentiviral em suspensão sem SFB apresentou resultados inferiores quando comparados com a produção em monocamada em meio suplementado com SFB. Por meio de Design de Experimentos (DoE) - Box-Behnken foi realizada uma otimização do protocolo de transfecção. Os resultados mostraram que a melhor condição envolveu a utilização de 1 μg de DNA/106 células, 1x106 células/mL e proporção PEI:DNA de 2,5:1. Este protocolo foi otimizado com a adição de Butirato de Sódio a 5mM e a produção obtida foi maior, em média, 1,5x105 partículas/mL. No presente trabalho foi possível realizar a otimização do protocolo de produção de partículas lentivirais em suspensão em meios livres de SFB mantendo as mesmas funcionais e capazes de induzir resposta antitumoral in vitro.Lentiviral vectors are widely used as vectors for gene modification in cell and gene therapies, such as dose employing CAR-T cells, due to their stability of integration in the cell genome, transduction efficiency and safety. The production of such vectors in monolayer serum-supplemented culture, traditionally used in academia and research centers, has a number of limitations; amongst them incompatibility with a more controlled large-scale production; being the suspension culture of cells in bioreactors desirable. The aim of this project was to produce lentiviral particles in scalable conditions, using cells adapted for serum-free suspension growth. Therefore, HEK293T cells adapted to serum-free suspension culture, were used to produce lentiviral particles that express the synthetic CAR protein anti-CD19 as well as IL-18 and GFP. The first step was to evaluate the production and functionality of lentiviral particles in monolayer serum-supplemented culture as a control condition. In this condition the average production was 8x106 particles/mL. The vector\'s functionality was assessed by modifying T cells to express the CAR molecule. The results indicated that the vector was capable of inducing antitumor response in T cells, which in turn were able to identify and kill CD19 positive cells in vitro. The adapted HEK293T cells had their kinetic and metabolic profile characterized, and satisfactory results were obtained with and without Cellboost supplementation. Nevertheless, the presence of Cellboost inhibited transient transfection and consequently the production of lentiviral particles. Even in the absence of Cellboost, serum-free suspension lentiviral production showed lower results when compared to monolayer serum-supplemented production. To solve that, an optimization of the transfection protocol was performed using Design of Experiments (DoE) - Box-Behnken. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1x106 cells/mL and PEI:DNA ratio of 2.5:1. This protocol was further optimized with the addition of 5mM Sodium Butyrate and resulted in an increase in the production, with an average of 1.5x105 particles/mL. In the present study, it was possible to optimize the protocol for the production of lentiviral particles in serum-free suspension culture, maintaining the functionality and capacity of inducing anti-tumor response in vitro

DataSheet_1_Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.docx

IntroductionNatural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary.MethodsIn this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence.ResultsWe demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model.ConclusionTogether with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.</p