CD4 and CD8 T-cell positive selection increases the robustness of the CD22 CAR T-cell manufacturing process

      CAR-T cell therapies targeting the surface markers CD19/CD22 have been very successful in the treatment of B-cell hematologic malignancies. One of the major challenges during CAR T cell manufacturing is low expansion and transduction of autologous CAR T cells. Poor performance is partly, if not entirely, due to the poor quality and quantity of cell components in the apheresis products from patients, particularly patients with high tumor burden. Flask adhesion and elutriation are often utilized as methods to remove monocytes and granulocytes, and anti-CD3/CD28 paramagnetic microparticles are used for T-cell enrichment and activation, but these approaches do not consistently result in an enriched CD3 T-cell population capable of efficient transduction and expansion. Often, residual tumor cells and monocytes remain within the culture and inhibit T-cell expansion. Here, we examine a total of 3 healthy donor samples and 2 B-ALL patient samples. We show that selection with anti-CD4/CD8 microparticles prior to stimulation and transduction with a CD22 CAR lentiviral vector results in increased T-cell expansion versus unselected cells. Moreover, a clinical sample from an ALL patient with high tumor burden that failed to undergo significant expansion while on the CD19 CAR clinical trial regained the ability to proliferate after CD4/CD8 positive selection and transduction with the CD22 CAR (4.9 fold vs. 27 fold expansion). CD3 T-cell percent increased from 7.2% in the starting product of the clinical trial to 50.3% post selection. In addition, tumor cells were efficiently depleted from the starting product as indicated by low percentage of CD22+ cells (from 43% to 0.6%). There were no significant differences when comparing transduction efficiencies from CD4/CD8 selected versus unselected products in a healthy donor and patient sample. The final product viability was >90% for all products tested. Xenograft models using human NALM6 ALL cell line showed equivalent tumor clearance between selected and unselected T-cell from a healthy donor. In conclusion, purification of T-cells in the starting product using CD4/CD8 positive selection prior to stimulation and transduction significantly enhances expansion of CD22 CAR T cells, especially problematic patient samples with high blast content, and allows for a more robust and consistent manufacturing process.
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