We have generated donor-derived T-cell lines specific for CMVpp65 peptides for use in a phase I, dose escalation trial of adoptive immunotherapy. T-cells were sensitized by autologous monocyte-derived DCs loaded with a pool of 138 pentadecapeptides (15-mers), with 11 amino acid overlaps spanning the entire 561 amino acid sequence of the CMV protein pp65. The 138 pentadecapeptides were synthesized and the T-cells were sensitized under GMP conditions. In preclinical studies we have been able to generate CMVpp65 specific T-cell lines from each seropositive donor tested, irrespective of HLA genotype. During the culture period of 21–35 days, populations of T-cells specific for CMV-pp65 selectively expanded 200–300 fold, while T cells reactive against major or minor alloantigens were depleted.
Thirteen pts with persistent CMV viremia, refractory to at least 2 weeks of therapeutic doses of ganciclovir or foscarnet, have been enrolled: 3 pts at a T cell dose of 5×105/kg, 3 pts at 1×106 T cells/kg, and 7 pts at 2×106 T cells/kg. CMV specific cytototoxic T lymphocytes (CTLs) were generated from HLA-identical unrelated donors (3 pts) or from HLA-identical siblings (10 pts). Two pts received conventional transplants after non-myeloablative conditioning; 11 pts received myeloablative conditioning and T-cell depleted transplants. Pts were eligible if they had persistent CMV viremia despite 2 weeks' treatment with antiviral drugs or had toxicities precluding further treatment with antiviral agents. Prior to infusion, T cell specificity against CMV was confirmed by cytotoxicity, intracellular interferon gamma (IFN-g) production, and MHC-tetramer staining (if available). The HLA-restrictions, epitope specificities, and TCR Vβ repertoires of the T-cell lines were also characterized before infusion. Cells were also assayed to establish lack of alloreactivity, microbiological sterility, and low endotoxin levels. All CTLs demonstrated cytolytic activity against peptide-loaded autologous PHA blasts but no cytotoxicity against non-pulsed HLA-matched or peptide-pulsed HLA-mismatched target cells. The proportion of CMVpp65-specific CD8+ cells in the infused T-cells, measured by intracellular IFN-g or MHC tetramers, ranged from 2 – 20 % or 4 – 70%, respectively. Post infusion, an increase in the absolute lymphocyte count correlated with an increase in CMV-specific T-cell frequencies to levels as high as 14% of CD8+ cells. In one pt, the CTLs were monitored and persisted for more than 2 years (10% of CD8+ cells) after the infusion. Notably, the same pp65-derived epitopes and HLA-restrictions which characterized the infused CTLs were detected in the pt specimens post infusion. The same TCR Vβ repertoires of the CMVpp65-specific CTLs infused were also detected post infusion. Donors for three of the treated pts expressed HLA-A*0201 and HLA-B*0701 alleles. Epitope-specific T cells for the HLA-A*0201-restricted NLVPMVATV peptide and the B*0701-restricted RPHERNGFTV peptide were detected and monitored in pre and post infusion T-cell populations in these three pts. In all three pts, the B*0701 restricted RPHERNGFTV emerged as the dominant T-cell population.
All 13 pts tolerated the infusions well without acute toxicities. None developed symptoms of GvHD at the dose levels tested. Twelve of the 13 pts cleared CMV viremia by 2–8 weeks following the CTL infusions. One of the pts died six weeks after the CTL infusion of respiratory failure despite clearing CMV from blood and bronchial aspirates. Another pt who initially remained viremic following the CTL infusion was restarted on oral valganciclovir and subsequently cleared CMV viremia. Only one pt had persistent viremia and died of pneumonia 31 days after CTL infusion.
The results from this trial demonstrate that donor T cells, sensitized with this pool of overlapping CMV pp65 pentadecapeptides, are safe and clear CMV viremia resistant to standard therapy. A larger phase II trial for the treatment of persistent CMV viremia and CMV infections is currently ongoing at MSKCC.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.