Development of CAR-T Cell Therapy for B-ALL Using a Point-of-Care Approach

Abstract

<jats:title>Abstract</jats:title><jats:p>Recently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for <jats:italic>ex vivo</jats:italic> activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective <jats:italic>in vivo</jats:italic> against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 hours of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity <jats:italic>in vivo</jats:italic>. Our data suggests that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.</jats:p>

CAR T cells: Building on the CD19 paradigm

“…In particular, minimally manipulated CAR T cells kept in culture for less than 24 h have demonstrated some level of antitumor activities in animal models and in human subjects. The optimization of this approach could simplify CAR‐T manufacturing and has the potential to drastically decrease the overall cost [129, 130].…”

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VII. Present and future of technologies for production of CAR cell therapies

“…Stimulating and culturing T cells for long period of time can lead to terminal differentiation. To overcome this limitation, and in order to optimize the phenotype of therapeutic T cells, some groups started reducing the culturing time 13 , 14 or adding cytokines that can help retaining a more memory and stem phenotype. Such cytokines include, but are not restricted to, IL-7, IL-15 15 and IL-21.…”

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost

“…Their group showed that isolation of PBMCs and genetic modification can be obtained in 4 h, generating CAR-T cells with in vivo activity. Cells generated with this rapid point of care compatible protocol were shown to have the same potency of CAR-T cells generated following in vitro expansion in pre-clinical models [ 139 ], indicating that CAR-T cells could be generated with minimal manipulation at the site of treatment using low cost protocols, especially in the allogeneic setting.…”