Improving Immune Reconstitution After Cord Blood Transplantation Using Ex Vivo Expanded Virus-Specific T Cells: A Phase I Clinical Study

Abstract 224

Adenovirus(Ad), Cytomegalovirus(CMV) and Epstein-Barr virus(EBV) frequently cause severe morbidity and mortality in patients(pts) after stem cell transplantation (SCT) and cord blood transplantation(CBT). We have shown that adoptive immunotherapy with peripheral blood(PB) donor derived multivirus-specific Cytotoxic T Lymphocytes directed against Ad, CMV and EBV can effectively prevent and treat the clinical manifestations of these viruses after SCT. CBT, while less likely to cause GvHD than conventional SCT, is unlikely to provide passive transfer of virus-specific CTL, since CBTs come from virus-naïve donors. Here we report for the first time the transfer of CB-derived multivirus-specific CTL(cbmCTL) to CBT recipients to restore cellular immunity to Ad, CMV and EBV. The development of cbmCTLs for pts undergoing CBT requires the priming and extensive expansion of naïve T cells rather than the more limited and simple direct expansion of pre-existing memory T cell populations from virus-exposed donors. We hypothesize that cbmCTL, derived from naïve T cells, will be efficacious and persist in vivo. Our protocol uses an initial round of stimulation with autologous CB-derived dendritic cells transduced with a recombinant Ad5f35 vector containing a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65) in the presence of IL-7, IL-12 (CTEP-NCI) and IL-15. This is followed by 2 rounds of weekly stimulation with autologous Ad5f35pp65-transduced EBV-LCL in the presence of IL-15 or IL-2.

Seven cbmCTL cultures generated for clinical use contained a mean of 48% CD8+, and 36% CD4+ cells with a mean of 33% CD45RA-/CD62L+ central memory T cells. In ⁵¹Cr release and/or IFNg ELISPOT assays, cbmCTL lines showed specific activity against all viruses. We have treated 7 pts who received the 80% fraction of a fractionated CB unit followed by cbmCTLs generated from the remaining 20% fraction; two pts were treated on each dose level;5×10⁶/m²; 1×10⁷/m²; and 1.5×10⁷/m² while one pt has been treated with 2.5×10⁷/m² – dose level 4. Pts received cbmCTLs on days 63–146 after CBT (median: day 83). No early infusion-related toxicities or subsequent GvHD was observed. All pts engrafted neutrophils by day 30 (median: day 20) despite receiving only 80% of the CB unit.

Five of 7 pts had no initial infection or reactivation episodes, remaining free of CMV, EBV, and Ad from 2 months to 2 years post-CBT. Of the two remaining pts, pt 1 was transiently viremic for CMV pre-infusion and became highly viremic 4-weeks post-cbmCTL. The pt received a 2^nd dose of cbmCTLs and CMV DNA/antigen became undetectable in the PB within 16 days of the 2^nd dose and remains asymptomatic and virus free >2 yr post-CBT. Analysis of this pt's PB showed a rise in CMV-T cells even prior to cbmCTL #2, with a 31-fold expansion of CMV-T cells by 4 weeks after the initial CTLs. This pt also had AdV in his stool, which resolved without additional therapy. Shortly after CTL infusion, pt 4 had detectable EBV DNA in the PB that was controlled without additional antiviral therapy.

The transferred cells had long-term persistence, since T cell receptor(TCR) deep-sequencing (ImmunoSEQ) allowed us to track infused T cell clones (i.e. clones present in the infused cbmCTL but absent in peripheral blood before cbmCTL infusion) up to 1 year post-CBT in 5/5 pts tested.

In summary, none of the recipients of cbmCTL developed viral disease; in two pts with viral infections, the infections resolved without progression to disease, coinciding with the appearance of virus-specific T cells in peripheral blood. Hence, administration of cbmCTL to pts after CBT has so far been safe and can facilitate reconstitution of virus-specific T cells and control viral reactivation/infection in vivo.