TCR gene transfer, TCR SE, and TCR CE redirect T cells toward NY-ESO-1. (A) Illustration summarizing procedure and timeline for the production of CE (upper panel), SE (middle panel), and TCR transferred (TR; lower panel) NY-ESO-1–redirected T cells. CE: At day 0 T cells harvested from healthy donors are stimulated with cell-sized beads coated with anti-CD3/CD28 antibodies (cell/bead ratio = 3:1) and kept in culture with low doses of IL-7 and IL-15 (5 ng/mL each). Two days later, the gene encoding for the constant region of the α chain (TRAC) is permanently disrupted by ZFNs delivered through AdVs. Gene modified cells are identified as CD3^neg, sorted by magnetic selection at day 8, and subsequently transduced with an LV to express the NY-ESO-1_157-165 TCR α chain. T cells expressing the tumor-specific TCR α chain reexpress CD3 on cell surface and can thus be selected and restimulated with a cell/bead ratio of 1:10 (S2). The subsequent steps of TRBC ZFN delivery (to disrupt the β chain constant region gene of the endogenous TCRs), CD3^neg cell sorting, and NY-ESO-1_157-165 TCR-specific β chain transduction occur at days 23, 35, and 36, respectively. The final T-cell product, after 5 to 6 weeks of cell manipulation, is a population of CE T cells fully and permanently redirected toward the NY-ESO-1 antigen. SE: After stimulation (S1), superimposable to that of the CE protocol, the TRAC gene is permanently disrupted by ZFNs delivered through AdVs or mRNA electroporation. CD3^neg cells are sorted and at day 9, transduced with an LV encoding the NY-ESO-1_157-165 TCR α and β chain genes. SE CD3^pos T cells, obtained in 2 weeks within S1, although still bearing the endogenous β TCR chains, are completely devoid of their endogenous TCR repertoire. TR: Two days after S1, activated T cells are transduced with an LV encoding for the NY-ESO-1_157-165 TCR α and β chains. Transduced cells are subsequently sorted either by Vβ13.1 or by NY-ESO-1_157-165 dextramer. (B) The efficiency of each manipulation step used in the CE, SE, and TR protocols was determined by flow cytometry on 4 healthy donors as follows: (i) transduction efficiency of the LV encoding for the NY-ESO-1_157-165 TCR (NY-ESO-1 α and β chains) for transferred (upper left panel) and SE (upper right panel) cells was determined by the quantification of NY-ESO-1 dextramer^pos and CD3^pos cells respectively; (ii) TRAC ZFN activity by AdV (orange dots) or mRNA electroporation (white dots) was measured by quantification of CD3^neg cells (lower left panel); (iii) transduction efficiency of LV encoding for the single NY-ESO-1 TCR α chain was determined by quantification of CD3^pos cells; (iv) TRBC ZFN adenoviral activity was measured by the quantification of CD3^neg cells; and (v) transduction efficiency of LV encoding for the single NY-ESO-1 β chain was assessed by quantifying CD3^pos cells (lower right panels). (C) Fold increase in the number of NY-ESO-1–redirected TR, SE, and CE T cells at the end of the manipulation procedure. Means and standard error of the mean (SEM) shown. *P = .013.