Safety and Clinical Efficacy of Rapidly-Generated Trivirus-Directed T Cells After Allogeneic Hematopoietic Stem Cell Transplant

We have previously demonstrated that small numbers of ex vivo-expanded, trivirus-specific T cells targeting EBV, CMV, and Adv are safe, effective and protective in vivo. However, broader implementation is limited by the need for infectious virus/vector, and prolonged (8-12wks) and complex manufacture, while antigenic competition limits extension to additional viruses. We now evaluate whether T-cell lines manufactured using methods that exclude viral components and utilize simplified manufacturing technology can be clinically effective. With NHLBI-PACT support, 29 clinical-grade rCTL lines have been generated. From an initial 15x10⁶ PBMCs, we prepared a median of 214±88x10⁶ T-cells (range 100-420x10⁶) over 9-11 days using DCs nucleofected with DNA plasmids encoding immunogenic EBV (LMP2, EBNA1 and BZLF1), Adv (Hexon and Penton), and CMV (pp65 and IE1) antigens, and expansion with IL4+7 in G-Rex devices. The rCTL lines were polyclonal, comprising both CD4+ (33±3%) and CD8+ (60.5±3%) cells, that expressed activation and memory markers. Twenty lines generated from donors that were seropositive for all viruses demonstrated activity against all 3 targets - CMV (IE1: 359±100; pp65: 637±177 SFC/2x10⁵), EBV (LMP2: 217±60, EBNA1: 67±19 and BZLF1: 111±31) and Adv (Hexon: 265±74, Penton: 191±53) - while 9 lines generated from CMV seronegative donors demonstrated activity exclusively against EBV (LMP2: 197±70, EBNA1: 145±51 and BZLF1: 239±84) and Adv (Hexon: 271±96, Penton: 254±90). None of the T-cell lines reacted against unmodified recipient cells. To date we have administered these lines to 10 allogeneic HSCT recipients at doses ranging from 0.5-2x10⁷/m² as treatment for CMV (n=3), Adv (n=2), EBV (n=2), EBV+Adv (n=1), and CMV+Adv (n=2). One patient developed a skin rash 2 weeks post-rCTLs but no other toxicity have been observed. Eight treated patients, including one with a biopsy-proven EBV lymphoma and the 3 patients with double reactivations, had complete clinical responses to rCTL, which corresponded with an increase in the frequency of virus-specific T-cells detected in peripheral blood. For CMV we saw an increase from a median of 0.5 to 96 and 1 to 277 SFC/4x10⁵ IE1 and pp65-specific T cells, respectively 3-6wks post-infusion; for Adv an increase from mean 0.5 to 137 and 0.5 to 99 SFC/4x10⁵ Hexon and Penton-specific cells, respectively, and for EBV an increase from 2.8 to 227, 1.5 to 39, and 1 to 188.5 SFC/4x10⁵ EBNA1, LMP2, and BZLF1-specific T-cells, respectively. Two patients failed to respond to rCTLs; one with a 3 year history of persistent CMV colitis and one with elevated EBV DNA; both had high pre-existing virus-specific T-cell precursors. rCTLs have been safe and effective in 80% of treated patients and have the potential to increase the availability of cell products for HSCT recipients. We are currently extending this platform to additional viruses.