Adoptive Immunotherapy with Rapidly-Generated Multivirus-Specific T Cells Against AdV, EBV, CMV, HHV6 and BK after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections remain a significant cause of morbidity and mortality after allogeneic stem cell transplantation (HSCT). We now report on the outcomes of 67 patients infused with either donor-derived (n=21) or third party "off the shelf" (n=46) virus-specific T cells (VSTs) with activity against 5 common post-transplant viruses (EBV, CMV, Adv, BK, HHV6).

We generated 84 clinical-grade VST lines by stimulating 30x10⁶ PBMCs with overlapping peptide libraries spanning Adv (Hexon, Penton), CMV (pp65, IE1), EBV (LMP2, EBNA1, BZLF1), BKV (Large T, VP1) and HHV6 (U11, U14, U90) antigens. After a 9-11 day expansion phase in a G-Rex device in the presence of activating cytokines, a mean of 399x10⁶ T cells (range 89-1006x10⁶) were harvested. The lines were polyclonal, comprising both CD4+ (60±2%) and CD8+ (31±2%) cells and expressed central CD45RO+/CD62L+/CCR7+ (38±3%) and effector memory markers CD45RO+/CD62L-/CCR7- (10±1%). VST specificity was dependent on the donor's prior exposure to viruses; thus, 81/84 lines had activity against Adv (Hexon: 384±44; Penton: 293±50 SFC/2x10⁵), 50/84 against CMV (IE1: 294±52; pp65 1009±194), 66/84 against EBV (LMP2: 153±26; EBNA1: 132±17; BZLF1: 91±25), 53/84 against BK (Large T: 134±22; VP1: 158±29) and 57/84 against HHV-6 (U90: 111±25; U14: 94±13; U11: 46±8).

Twenty one allogeneic HSCT recipients were infused with donor-derived VSTs in a dose escalation study with informed consent; 4 on DL1 (5x10⁶/m²), 4 on DL2 (1x10⁷/m²) and 13 on DL3 (2x10⁷/m²). Four patients received the cells prophylactically and remained free of viral infections up to 3 months post-infusion. The remaining patients were infused for one or more active infections. There were 4 cases of Grade I-II GvHD, of which only one required treatment with steroids. Based on viral load measurements and symptom assessment a single VST infusion successfully controlled active infections associated with all our targeted viruses (ORR 95%): CMV (7 CR, 1PR); EBV (7 CR, 1PR); Adv (6 CR); HHV6 (5 CR, 1PR, 1NR) and BKV (8 CR, 2 PR, 1 NR). Overall, we used VSTs to treat 7 cases of viral disease- EBV-PTLD (n=1), HHV-6 encephalitis (n=1), BKV hemorrhagic cystitis (HC) (n=5). The infused cells successfully cleared the EBV-PTLD (without rituximab) and HHV6 encephalitis, while all patients with BKV HC had marked improvement or resolution of symptoms post-VSTs.

Based on the safety and efficacy profile of VSTs we extended our approach to third party recipients. To date we have treated 46 patients, all of whom had drug-refractory infections. Patients received 1-3 doses (fixed dose - 2x10⁷ cells/m2) with 3rd party VSTs matching at one to six of eight HLA alleles. Despite the HLA disparity the cells have proven safe with three cases of de novo grade I-II GvHD documented. Third party VSTs successfully controlled active infections in 42 of 45 evaluable patients (ORR 94%): CMV (13 CR, 5 PR, 1 NR); EBV (2 CR); AdV (7 CR, 2 NR); BKV (5 CR, 14 PR); and HHV-6 (4 PR). Of note, 30 of 46 patients were treated for viral disease - CMV colitis (n=3), CMV encephalitis (n=1), EBV-PTLD (n=1), AdV respiratory tract infection (RTI) (n=2), AdV RTI and enteritis (n=1), AdV HC (n=1), HHV-6 encephalitis (n=2), BKV HC (n=17), BKV nephritis (n=2). All patients with BK-HC, 2 of 3 patients with CMV colitis, and the patients with CMV encephalitis, AdV RTI, enteritis and HC, and HHV-6 encephalitis had marked improvement or resolution of symptoms within 6 weeks following VST treatment. In more than 50% of responders (n=24) we detected an increase in the circulating frequency of VSTs post-infusion, which were confirmed to be 3^rd party VST origin in 14 and remained detectable for up to 12 weeks post-infusion.

Overall our experience with adoptively-transferred VSTs demonstrates not only the safety profile of the cells, even when administered as a 3^rd party product, but also the efficacy of the infused cells in patients with up to 4 simultaneous/sequential infections, and HSCT recipients with virus associated disease.