GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL.

BACKGROUND: Pre-clinical studies suggest Xcellerated T Cells have the potential to produce a potent anti-tumor effect, restore broad immune function and reduce the risk of infectious complications in patients with CLL. Unlike other cancer settings, T cells constitute only a small fraction of CLL patients' PBMC. To generate large numbers of Xcellerated T Cells of high purity from CLL patients' PBMC, a reproducible, streamlined and cost-effective good manufacturing process (GMP) is required. METHODS: The 10-L volume Wave Bioreactor-based Xcellerate III Process using Xcyte Dynabeads in a single custom 20-L Cellbag container was adapted, qualified and implemented for GMP operations. RESULTS: For n=17 CLL patients, starting with approximately 1.34 x 10(9) CD3+ T cells at 6.8+/-7.5% purity in the PBMC leukapheresis products, using the 10-L volume Wave Bioreactor-based Xcellerate III Process, it was feasible to manufacture 137.0+/-34.3 x 10(9) Xcellerated T Cells at 98.5+/-1.0% CD3+ T-cell purity. An average 400-fold clearance of malignant B cells was documented during the manufacturing process. The Xcellerated T Cells produced from the Xcellerate III Process exhibited high in vitro biologic activity and have their T-cell receptor repertoire restored to a normal diversity. In-process T-cell activation was reproducibly robust, as measured by increase in cell size, up-regulation of CD25 and CD154 expression and the secretion of IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha. DISCUSSION: A low-volume, high-yield bioreactor-based process has been developed, qualified and implemented for the reproducible, GMP manufacture of high purity, biologically active Xcellerated T Cells for the treatment of CLL patients in clinical trials.

Selective pharmacologic inhibition of murine and human IL-12-dependent Th1 differentiation and IL-12 signaling.

We have previously shown that lisofylline (LSF) inhibits murine Th1-mediated disease in vivo by blocking IL-12-induced differentiation of Th1 cells. The cellular and molecular mechanisms underlying this inhibition were further explored by testing LSF in several IL-12-responsive model systems in vitro. IL-12-dependent Th1 differentiation was abrogated by LSF and yielded effector T cells that were deficient in proinflammatory cytokine secretion, including IFN-gamma, IL-2, and TNF-alpha. The diminished Th1 phenotype resulted from both a lower frequency of IL-12-derived Th1 clones and a reduced capacity of individual clones to secrete IFN-gamma due to lower levels of IFN-gamma mRNA. The arrest in Th1 development resulted from a blockade of IL-12 signaling that preceded the Th0 to Th1 transition. Thus, LSF blocked IL-12-enhanced IFN-gamma production in anti-CD3-stimulated T cells and prevented IL-12-mediated repression of the transcription factor GATA-3. Lisofylline also inhibited IL-12-induced increases in STAT4 tyrosine phosphorylation, but did not block TCR signaling or inhibit acquisition of IL-12 responsiveness. These findings were extended to show that LSF also inhibits IL-12-dependent responses in human T cells. LSF, which has one asymmetric chiral center, was selectively inhibitory for IL-12 signaling compared with its S-enantiomer (1501-S) and the oxidized side chain analog, pentoxifylline. The results suggest that LSF may be useful as a modulator of Th1-mediated disease in humans.