DEVELOPMENT OF CAR-T CELL THERAPY FOR B-ALL USING A POINT-OF-CARE APPROACH

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  • Presentation type: Doutorado
  • Track:
  • Keywords: CAR-T cells; point-of-care; Sleeping Beauty; Immunotherapy;
  • 1 INSTITUTO NACIONAL DE CÂNCER (INCA)
  • 2 Fundação Oswaldo Cruz (FIOCRUZ)

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Abstract

INTRODUCTION AND OBJECTIVE: CD19-targeted CAR-T cell immunotherapy is now an approved treatment for B cell leukemias and lymphomas. Current protocols for producing CAR-T cells use viral vectors for genetic modification and take 12-15 days to expand sufficient numbers of T cells, constituting an important bottleneck for the widespread use of this therapy. The development of non-viral, plasmid-based systems like Sleeping Beauty (SB) transposons, coupled with the emergence of large scale, closed electroporation devices, present the opportunity of generating high numbers of CAR-T cells in a short period of time and transforming this in a point-of-care (POC) therapy.
MATERIAL AND METHOD: In this work, mononuclear cells were isolated using Ficoll and electroporated using Nucleofector IIb electroporator combined with plasmids encoding 19BBz CAR (pT3 SB transposon backbone) and SB100x transposase. Cells were rested for 24h and used for in vitro and in vivo experiments. The phenotype was assessed by flow cytometry. The in vitro cytotoxicity assay was performed using Calcein-AM dye on target cells incubated with different ratios of effector cells. 8-12-week-old-female-NSG were injected iv. 5x10e6 RS4;11 GFP or 10e5 Nalm-6 GFP and after 3 days were treated with different doses of recently electroporated CAR-T cells.
RESULTS AND CONCLUSION: CAR expression on day 1 following electroporation ranged between 5-15% and in vitro cytotoxic activity against RS4;11 or Nalm-6 CD19+ B cell precursor leukemia lines was low or absent. NSG mice engrafted with RS4;11 and treated with 1x105 19BBz+ cells showed improved survival when compared to mice treated with mock electroporated cells. POC approach was also efficient against Nalm-6, where 19BBz+CAR-T cells (7x10e5 per mice) improved the survival of mice. In both models, decreased tumor burden in blood and spleen was observed in mice treated with 19BBz. Head to head comparison of 19BBz cells used in POC approach or expanded for 8-12 days in vitro showed similar antitumor activity in vivo against RS4;11 cells, leading to equivalent improvements in mice survival. In summary, we show that the POC strategy is a viable approach for the use of CAR-T cells, obviating the need for T cell expansion while having the potential to greatly decrease the total cost and time of CAR-T cell therapy manufacturing.

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Author

Luiza Abdo

Olá João, muito obrigada pela pergunta. Nesse primeiro momento avaliamos apenas os tumores de LLA-B CD19+. Foram duas linhagens com diferentes comportamentos in vivo, RS4;11 e NALM-6. Estamos começando um projeto em tumores sólidos para utilizarmos células CAR-T anti-HER-2. Vamos trabalhar com tumores de ovários e pâncreas. Nesses experimentos, não observamos nenhum efeito adversos nos camundongos tratados, apenas crescimento tumoral. E por fim, pretendemos sim continuar esse estudo para pacientes humanos. Já começamos a avaliar modelos pré-clinicos porém, utilizando o protocolo tradicional de expansão das células . É possível ver esses dados na apresentação do pós-doutorando Leonardo Silva. O uso da abordagem de modificação gênica SB se mostra mais acessível que os vetores virais usualmente utilizados e já foi utilizando em Clinical trial, desmonstrando ser segura e eficaz. Isso é um ponto positivo no aspecto de um país em desenvolvimento. O caminho para aprovação da abordagem POC pode ser um pouco mais longo que o tradicional, mas temos pretensão de seguir sim, pois acreditamos que essa metodologia pode favorecer ainda mais os pacientes, pois o custo tende ser muito menor. Como sabemos, há várias barreiras na ciência brasileira, mas estamos caminhando para seguir com essa terapia, que acreditamos que pode trazer inúmeros benefícios. Obrigada mais uma vez, qualquer dúvida estou à disposição. Abraço