Prophylactic Infusion of Multi-Antigen Specific T-Cell Products to Prevent Complications after T Cell Depleted Allogeneic Stem Cell Transplantation — a Phase I/II Study
Authors: Roex Marthe C.J. , Balen Peter van , Germeroth Lothar , Hageman Lois , van Egmond Esther , Veld Sabrina A.J. , Hoogstraten Conny , van Liempt Ellis , Zwaginga Jaap J. , de Wreede Liesbeth C. , Einsele Hermann , Veelken Hendrik , Halkes Constantijn J.M. , Jedema Inge , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed.
HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for inclusion. Donor T cells directed against HLA-A2-restricted peptides of CMV, EBV, AdV, and TAA NY-ESO-1, WT-1, RHAMM, PRAME and proteinase 3 were simultaneously isolated under GMP conditions in 1 day, using MHC I-Streptamers and CliniMACS selection out of the naïve and memory T cell compartment from 2*109 PBMC. Depending on patient/donor HLA-typing, additional MHC I-Streptamers targeting viral peptides in HLA-A1, -A24, -B7, or -B8 were added to the procedure, as well as the HLA-A2/HA-1h MHC I-Streptamer in case of MiHA disparity in the GVL direction. The incidence of GVHD, mortality, disease relapses and viral reactivations was monitored until 6 months after alloSCT. In addition, follow-up samples were stained with tetramers to analyze in vivo reconstitution of target-Ag specific T cells.
Products were generated for 27/28 included patients that showed stable engraftment after TCD alloSCT; 1 patient died early after alloSCT. All donors were EBV+, 14/27 donors were CMV+. 26/27 products were successfully generated and contained a median of 5.2*106 cells (range 0.4 - 26.5*106) with a median purity of target-Ag specific T cells within the CD3+ compartment of 80.4% (range 46.0-99.9). Target-Ag specific T cells consisted for 99% of virus-specific memory T cells, while the remaining 1% included TAA, MiHA and naïve virus-specific T cells. 24 patients received their product (median 58 days after alloSCT; range 51-107) without infusion related complications; in 2 patients the infusion was cancelled due to acute skin GVHD on the day of infusion. During follow-up, 1 patient experienced skin GVHD requiring immunosuppressive therapy. One patient died due to complications of a nephrotic syndrome, probably unrelated to product infusion. Four patients showed disease relapse and were treated accordingly; coinciding expansion of TAA- or MiHA-specific T cells was not observed. None of the patients experienced AdV reactivations; in 2 patients AdV-specific T cells expanded after infusion. Four patients had detectable EBV loads; in one of these patients the EBV reactivation progressed to EBV-PTLD and he was treated with rituximab followed by DLI. In 2 patients, the rise in EBV load led to expansion of EBV specific T cells in vivo. 4/5 CMV+ patients with a CMV- donor had ongoing CMV reactivations at the moment of product infusion, CMV specific T cells could already be detected just before product infusion and reactivations were cleared within 7-84 days. Of the 8 CMV+ patients with a CMV+ donor, 3 patients remained free of CMV reactivations, 4 had already a reactivation at the moment of product infusion and 1 patient developed a CMV reactivation during follow up. One patient had progressed to CMV pneumonia within 2 weeks after infusion, requiring ganciclovir. In all 5 CMV reactivating patients, T cells directed against 2-3 CMV-specific epitopes expanded significantly and viral loads declined. TCR sequencing of the CDR3 region illustrated in vivo persistence and expansion of target-Ag specific T cells present in the product.
In conclusion, we have shown that MHC I-Streptamer isolation based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is safe and feasible. Moreover, efficacy of the prophylactic infusion is illustrated by expansion of target-Ag specific T cells in patients coinciding viral reactivations and the prevention of viral complications in the majority of patients between TCD alloSCT and DLI.

Disclosures
Germeroth: Juno Therapeutics: Employment, Equity Ownership.