Residual TH9402 concentrations are minimal and just higher than the background of the untreated control (N = 3). light energy at 5 106 cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100?000, while third-party responses remained higher than 1:10?000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte D-γ-Glutamyl-D-glutamic acid transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation. Introduction Overall survival, following allogeneic stem cell transplantation (SCT) for malignant diseases has steadily improved, largely due to reduced transplantation-related mortality.1 In contrast, risk-stratified relapse rates have not changed significantly over the past 3 decades. 2 Further improvements in SCT outcome thus await improved control of the malignant disease. One approach is to exploit the unique graft-versus-leukemia (GVL) effect of alloreacting donor immune cells.3C5 Currently GVL is limited by the fact that strategies to prevent graft-versus-host disease (GVHD) with immunosuppression or T-lymphocyte depletion tend to compromise the GvL effect. While T cellCmediated GVHD and GVL depend on similar mechanisms, the antigenic overlap between normal tissue targets of GVHD and between leukemic tissues and normal tissues is not complete. As a consequence, different T-cell populations can distinguish myeloid from lymphoid tissues as well as leukemic from normal cells in vitro.6C9 Thus it should be possible to selectively eliminate GVHD-causing donor lymphocytes from allografts while sparing the valuable T cells exerting GVL and beneficial antimicrobial responses. This approach, usually referred to as selective lymphocyte depletion (SD) or selective allodepletion, uses patient-derived antigen-presenting cells (APCs) for stimulation of donor T cells in an ex vivo coculture. Alloactivated donor lymphocytes are then removed by virtue of their activation status.10 SD has been achieved using D-γ-Glutamyl-D-glutamic acid immunomagnetic beads or immunotoxin specific for surface markers of early T-cell activation such as CD25,7,9,11C19 CD69,15,19,20 CD71,19 CD137,21 or HLA-DR,19 sorting of nonactivated22 or nonproliferating cells using dye-dilution techniques,23 apoptosis induction24,25 and photodepletion (PD)26,27 targeting the impaired ability of activated T cells to efflux a phototoxic rhodamide-like dye (TH9402) due to changes in their multidrug-resistance pump p-glycoprotein (MDR1). The latter approach was shown to be feasible in an HLA-mismatched, small-scale system both in mice26 and humans.27 Clinical data on SD are limited, but there are promising results from 3 clinical trials using an antiCCD25-immunotoxin for removal of alloactivated T cells.28C30 We found that SD using an anti-CD25-immunotoxin may protect against acute GVHD (aGVHD) both by removal of alloactivated T cells and by allowing regulatory CD4+ T cell (Treg) reconstitution.30,31 The occurrence of residual aGVHD in our series of selectively allodepleted transplantations was associated with low donor Treg numbers31 and poor depletion efficacy.30 Inefficient TFR2 allodepletion could be due to a down-regulation of CD25 antigen during the coculture period, allowing some alloactivated cells to escape the depletion process. Here, we aimed to improve the efficiency and reliability of D-γ-Glutamyl-D-glutamic acid SD using a TH9402-based PD method as an alternative strategy to surface marker targeting. We worked with clinical-scale cell volumes under identical conditions used for current Good Manufacturing Practice (cGMP). The use of expanded lymphocytes as antigen-presenting cells (APCs) allowed the generation of large numbers of allodepleted T cells. The established PD process was capable of highly efficient removal of alloreactive lymphocytes from mismatched and matched cocultures and maintained desirable third-party responses including antiviral and antibacterial responses Methods Human subjects Protocols that included procedures for leukapheresis collections from patients with hematologic malignancies, healthy sibling donors, and unrelated, healthy volunteers were approved by the National Heart, Lung, and Blood Institute (NHLBI) Institutional Review Board. Informed consent was obtained from all patients, healthy sibling donors, and healthy volunteers in accordance with the Declaration of Helsinki. Current Good Manufacturing Practice Stimulator generation, coculturing, and photodepletion procedures were performed under conditions mimicking cGMP. Using automated instruments with sterile disposable kits, bags, and sterile connecting devices for cell transfer (Terumo, Eschborn, Germany) a semiclosed culture and treatment system was used in anticipation of a clinical trial. All reagents used either were drugs approved by the US Food and Drug Administration (FDA) or had a certificate of analysis certifying safety and characterization requirements. Final cellular product safety testing including sterility, and endotoxin and mycoplasma assays were performed and found to be within acceptable limits for all products. Stimulator generation Ex vivoCexpanded.
