Manufacture of CD22 CAR T cells following positive versus negative selection results in distinct cytokine secretion profiles and γδ T cell output

Abstract

Chimeric antigen receptor Tcells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, Tcell selection, removes contaminating cell types that can potentially suppress Tcell expansion and transduction. While positive selection of CD4/CD8 Tcells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher invitro interferon-γ and IL-2 secretion but a lower invitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial Tcell selection process in clinical CART manufacturing.