Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8+ cell depletion and subsequent CD25high enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13–14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 109 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4+CD25highCD127lowFOXP3+ cells that had similar in vitro immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg)

G-CSF in Healthy Allogeneic Stem Cell Donors

Mobilization of peripheral blood stem cells (PBSC) in healthy volunteers with granulocyte colony-stimulating factor (G-CSF) is currently carried out at many institutions worldwide. This report presents the experience of the Dresden center regarding donor evaluation and mobilization schedule. Data regarding efficacy, short- and long-term safety of G-CSF treatment gained from 8290 PBSC collections in healthy donors are outlined. These results are discussed against the background of the available evidence from the literature. Although established as a standard procedure, G-CSF application to allogeneic donors will always be a very delicate procedure and requires the utmost commitment of all staff involved to ensure maximum donor safety. (PBSC) donation does not require hospitalization and is generally assumed to be less physically demanding for the donor. However, application of mobilizing agents is stringently required for successful HSC mobilization. The standard substance, which is almost exclusively used in healthy donors worldwide, is recombinant human granulocyte colony-stimulating factor (rhG-CSF). Two preparations – filgrastim and lenograstim – are available and have been approved for PBSC mobilization for about 15 years in Germany. Currently, more than 20,000 healthy donors worldwide receive rhG-CSF for PBSC mobilization every year [7]. At the Dresden University Hospital, PBSC collections have been performed since 1996. In the two collection facilities associated with the university hospital, 8,290 allogeneic PBSC collections from 8,005 donors (i.e. 285 second collections) have been documented in a database up until May 2012. This paper presents the data of our own group, and summarizes the current knowledge regarding the short- and long-term effects of G-CSF treatment in healthy stem cell donors

Leukocyte Depletion by Therapeutic Leukocytapheresis in Patients with Leukemia

Hyperleukocytosis is a complication of various leukemias and can result in life-threatening leukostasis. Critical white blood cell (WBC) counts are conventionally defined as higher than 100 × 109/l in acute myeloid leukemia and > 300 × 109/l in acute lymphatic leukemia and other leukemic disorders (e. g. chronic myeloid leukemia). Leukocytapheresis is a therapeutic tool to reduce leukocyte counts in patients with symptomatic or threatening leukostasis until induction chemotherapy works. In patients with temporary contraindications against cytotoxic drugs, e.g. during pregnancy, leukocytapheresis can be used as a bridging therapy until conventional chemotherapy can be started. Therapeutic leukocytapheresis should be performed in specialized centers by experienced, well-trained staff. Thorough monitoring of the patients is extremely relevant. During a single procedure, WBC count can be reduced by 10–70%. Treatment should be repeated daily and can be discontinued when the symptoms of leukostasis have been resolved and/or leukocyte counts have fallen below the critical thresholds. There are no prospective studies evaluating the clinical efficacy of therapeutic leukocytapheresis in patients with hyperleukocytosis. It can be concluded from retrospective studies that leukocytapheresis might have some beneficial effect in early morbidity and mortality of patients with newly diagnosed AML but has no influence on overall long-term survival. Induction chemotherapy is the most important treatment in these patients and must never be postponed.Leukozytose ist eine Komplikation verschiedener Leukämien und kann zur lebensbedrohlichen Leukostase führen. Als kritische Leukozytenzahlen gelten im Allgemeinen Werte über 100 × 109/l bei akuten myeloischen Leukämien und über 300 × 109/l bei akuter lymphatischer Leukämie und anderen Leukämieformen (z. B. chronisch-myeloische Leukämie). Mittels therapeutischer Leuko zytapherese können pathologisch erhöhte Leukozytenwerte bei Patienten mit symptomatischer oder drohender Leukostase reduziert werden, bis die Wirkung der Induktions-Chemotherapie einsetzt. Bei Patienten mit vorübergehenden Kontraindikationen gegen Zytostatika, wie z.B. in der Schwangerschaft, dient die Leukozytapherese zur Überbrückung des Zeitraums, bis die konventionelle Chemotherapie begonnen werden kann. Leukozytapheresen sollten nur in spezialisierten Zentren von erfahrenem, geschultem Personal durchgeführt werden. Eine sorgfältige Überwachung der Patienten ist von besonderer Bedeutung. Während einer Behandlung kann die Leukozytenzahl um 10–70% reduziert werden. Die Behandlung sollte täglich wiederholt werden, bis die Leukostasesymptomatik abgeklungen bzw. die Leukozytenzahl unter die kritische Interventionsschwelle abgefallen ist. Es mangelt an prospektiven, randomisierten, kontrollierten Studien, die den klinischen Effekt der therapeutischen Leukozytapherese bei Patienten mit Leukostase evaluieren. Retrospektive Studien lassen auf eine therapeutische Wirksamkeit der Leukozytapherese hinsichtlich Frühmorbidität und –mortalität bei Patienten mit neu diagnostizierter AML schließen. Ein Einfluss dieser Therapie auf das Gesamtüberleben von AML-Patienten konnte nicht nachgewiesen werden. Die entscheidende Therapie für diese Patienten ist die Induktions-Chemotherapie, die deshalb auch keinesfalls verzögert werden sollte.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Salvage treatment with plerixafor in poor mobilizing allogeneic stem cell donors: results of a prospective phase II-trial

We conducted a prospective clinical trial to investigate the safety and efficacy of plerixafor (P) in allogeneic peripheral blood stem cells (PBSC) donors with poor mobilization response to standard-dose granulocyte colony-stimulating factor (G-CSF), defined by <2 × 1