ALBERT

Description: Key Points Pathogen-inactivated platelets were noninferior in preventing bleeding only in intention-to-treat analysis. In contrast to animal models, alloimmunization could not be prevented when using pathogen-inactivated platelets.

Description: 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.

Description: β2-Integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor–related protein (LRP) as a new partner for β2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against β2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the αMβ2 complex and the αM and αL I-domains (Kd, app ≈ 0.5 μM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and αLβ2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of αLβ2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of αLβ2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of β2-integrin–dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte function.