Abstract
Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. Ex vivo cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. TCM and TEM were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a TN or TCM phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that ex vivo cell expansion leads to reduced numbers of TN, TSCM, and TEFF cells, while TCM cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced ex vivo expansion may yield CAR T-cell products with increased persistence in vivo.
Highlights
Adoptive T-cell transfer (ACT) immunotherapy is a field in continuous expansion especially during the last three decades
Most of the approaches explored so far are based on the use of autologous Chimeric Antigen Receptor (CAR) T-cells, thereby requiring the production of a personalized product for each patient
We have previously published the development of our own CAR19 product and the establishment of a CAR T-cell production protocol and infrastructure, based on the use of CliniMACS Prodigy, a semi-automated closed system by Miltenyi [37]
Summary
Adoptive T-cell transfer (ACT) immunotherapy is a field in continuous expansion especially during the last three decades. ACT involves ex vivo expansion of tumor-specific cells and reinfusion into the patient. Among these therapies, the use of Chimeric Antigen Receptor (CAR) T-cells for the treatment of several hematologic malignancies has shown unprecedented efficacy rates. CARs are chimeric proteins composed of an extracellular region responsible for binding to a particular antigen and an intracellular region that promotes T-cell cytotoxic activity and proliferation. The scFv-derived region results in a MHCindependent interaction of the CAR with its ligand. This scFv is combined with one or more intracellular co-stimulatory domains (usually CD28 or 4-1BB) and a pro-activator cytotoxic domain (CD3ζ) [4,5,6]
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